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Table of Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Gene Expression Analysis . . . . . . . . . . . . . . 2
2.1. RiboQuant Multi-Probe Ribonuclease
Protection Assay System . . . . . . . . . . . . . . 2-6
2.1.1. Custom RiboQuant RNase
Protection Assays . . . . . . . . . . . . . . . . 7
2.2. Atlas Arrays . . . . . . . . . . . . . . . . . . . . . 8-10
3.2. Caspases . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
BD, BD Logo and all other trademarks are the property of Becton, Dickinson and Company. 2002 BD
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Abbreviations
AFC
AMC
APC
Apps.
DsRed
E
EBS
EGFP
EYFP
FA
FBS
FC
FITC
fmk
GSH
Hu
IF
IHC(F)
IHC(P)
IP
IVK
LM-PCR
Ms
MTC
NA/LE
NES
NLS
NO
PI
PE
pNA
PS
R
RPA
RXNS
SF
TUNEL
WB
7-amino-4-trifluoromethy
coumarin
7-amino-4-methoxy coumarin
Allophycocyanin
Applications
Discosoma sp. Red Fluorescent
Protein
ELISA
Epitope Blocking Studies
Aequorea victoria Enhanced
Green Fluorescent Protein
Aequorea victoria Enhanced
Yellow Fluorescent Protein
Functional Assays
Fetal Bovine Serum
Flow Cytometry
Fluorescein isothiocyanate
Fluoromethylketone
Glutathione
Human
Immunofluorescence
Immunohistochemistry (Frozen)
Immunohistochemistry (Paraffin)
Immunoprecipitation
In Vitro Kinase Assay
Ligation-mediated polymerase
chain reaction
Mouse
Multiple Tissue cDNA
No Azide/Low Endotoxin
nuclear export signal
nuclear localization signal
Nitric oxide
Propidiumiodide
Phycoerythrin
p-Nitroanilide
Phosphatidylserine
Rat
Ribonuclease Protection Assay
Reactions
Spectrofluorometry
terminal deoxynucleotidyl
transferase-mediated dUTP
nick-end-labeling
Western Blot
Introduction
Programmed cell death is a normal
physiological process which occurs
during embryonic development as
well as in maintenance of tissue
homeostasis. Kerr et al. originally
described two forms of cell death
which may occur in the absence of
pathological manifestations, necrosis and apoptosis1. The term apoptosis, from the Greek word for
falling off of leaves from a tree, is
used to describe a process in which
a cell actively participates in its own
destructive processes. The apoptotic
program is characterized by certain
morphological features. These
include changes in the plasma membrane such as loss of membrane
asymmetry and attachment, a condensation of the cytoplasm and
nucleus, and internucleosomal
cleavage of DNA. In the final
stages, the dying cells become fragmented into apoptotic bodies
which are rapidly eliminated by
phagocytic cells without eliciting
significant inflammatory damage to
surrounding cells. Necrosis, which
typically occurs as a result of cell
injury or exposure to cytotoxic
chemicals, is distinct from apoptosis
in both morphological and biochemical characteristics. Necrotic
cell death begins with swelling of
the cell and mitochondrial contents,
References
1. Kerr, J.F.R., A.H. Wyllie and A.R. Curie. 1972. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26:239-257
2. Majno, G. and J. Joris. 1995: Apoptosis, oncosis and necrosis. An overview of cell death. Am. J. Pathol.
146:3-16.
3. Kitumura, Y., Perry, P.J. Whitehouse and T. Taniguchi. 1998. Alteration of proteins regulating apoptosis, Bcl-2, Bcl-x,
Bax, Bak, Bad, ICH-1 and CPP32, in Alzheimers disease. Brain Res. 780:260-269.
4. Lorenzen, J., J. Thiel and R. Fischer. 1997. The mummified Hodgkin cell: Cell death in Hodgkins disease. Am. J. Pathol.
182:288-298
5. Lin, T., Brunner, B. B. Tietz, J. Madsen, E. Bonfoco, M. Reaves, M. Hufet and D.R.Green. 1998. Fas ligand-mediated
killing by intestinal intraepithelial lymphocytes. Participation in intestinal graft-versus-host disease. J. Clin. Invest. 101:5705747
6. Thomson, C.B. 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267:1456-1462.
7. Wang, L., G.R. Klimpel, J.M. Planas, H. Li and M.W. Cloyd. 1998. Apoptotic killing of CD4+ T lymphocytes in HIV-1
infected PHA-stimulated PBL cultures is mediated by CD8+ LAK cells. J. Virol. 241:169-180
www.bdbiosciences.com
hAPO-2
1
bcl-x
L,S
bcl-x
bfl1
bfl1
bik
bak
bik
bax
bak
bcl-2
bax
mcl-1
bcl-2
mcl 1
L32
PBMC
PBMC
Stimulated
Jurkat
Stimulated
Probe
Jurkat
GAPDH
hAPO-3
1
Caspase-8
FASL
FAS
FASL
FADD
FAS
DR3
FAP
DR3
FAF
TRAIL
TRAIL
TNFRp55
TRADD
RIP
TNFRp55
TRADD
L32
Stimulated
PBMC
PBMC
Jurkat
Stimulated
Probe
Jurkat
GAPDH
www.bdbiosciences.com
Day 1:
Probe Synthesis
RNA Preparation
Overnight Hybridization
Day 2:
Gel Preparation
Electrophoresis on Denaturing
Polyacrylamide Gel
Transfer to membrane
Chemiluminescent signal detection
Expose membrane to film
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Product Listing
Description
Size
Cat. No.
45004K
45013Z
45006Z
45012Z
45005Z
45010Z
45008Z
45009Z
45007A
45011Z
556850
556133
556126
556132
556125
556130
556128
556129
556127
556131
551917
45014K
45021Z
45015A
45022A
45019Z
45018A
45017Z
45016A
45020Z
556134
556141
556135
556142
556139
556138
556137
556136
556140
551918
45024K
45004K
45014K
556144
556850
556134
45014K
551917
556134
551918
hAPO-1b
hAPO-1c
hAPO-2b
hAPO-2c
hAPO-3
hAPO-3b
hAPO-3d
hAPO-4
hAPO-5
hAPO-5b
hAPO-5c
hAPO-6
10
10
10
10
10
10
10
10
10
10
10
10
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
transcription
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
reactions
45378P
45607P
45614P
45609P
45131P
45611P
45616P
45141P
45151P
45610P
45613P
45612P
556209
556233
556240
556235
556163
556237
557278
556164
556165
556236
556239
556238
10
10
10
10
transcription
transcription
transcription
transcription
reactions
reactions
reactions
reactions
45358P
45354P
45355P
45601P
556195
556191
556192
556227
mAPO-1
mAPO-2
mAPO-3
rAPO-1
www.bdbiosciences.com
BD Pharmingen
Product Listing
Template
Protected
hAPO-1b
Protected
hAPO-1c
Caspase-8
Granzyme B
Caspase-3
Caspase-6
Caspase-5
Caspase-2 (S)
Caspase-7
Caspase-1
Caspase-2 (L)
Caspase-9
-L32 112
GAPDH
406
361
321
283
227
202 (S)
181
163
150 (L)
127
--
Template
Template
Protected
hAPO-2b
96
Caspase-8
Caspase-4
Caspase-3
Caspase-6
Caspase-10a
Caspase-5
Caspase-2 (S)
Caspase-7
Caspase-1
Caspase-2 (L)
Caspase-9
L32
GAPDH
406
361
320
283
253
227
203 (S)
181
163
150
127
113
96
bcl-W
bcl-X (L)
bcl-X (S)
bfl-1
BID
bik
bak
bax
bcl-2
mcl-1
-L32
GAPDH
401
364
324
286
257
226
202
181
160
139
-113
96
Protected
Template
Protected
Template
Protected
hAPO-2c
hAPO-3b
hAPO-3
bcl-W
bcl-XL
bcl-XS
bfl-1
bad
bik
bak
bax
bcl-2
mcl-1
-L32 113
GAPDH
401
364 (L)
324 (S)
286
257
226
202
181
160
139
--
Template
96
Caspase-8
FasL
Fas
Fadd
DR3
FAP
FAF
Trail
TNFRp55
Tradd
Rip
L32
GAPDH
406
353
316
283
253
226
202
181
160
149
134
113
96
Protected
Template
Protected
hAPO-3d
Caspase-8
FasL
Fas
DCR-1
DR3
DR5
DR4
Trail
DCR-2
TNFRp55
Tradd
Rip
L32
GAPDH
Template
Caspase-8
FasL
Fas
CLARP
FAP
CRADD
Daxx
MADD
RIP
--L32
GAPDH
406
351
316
253
226
199
163
151
134
--113
96
hAPO-4
406
351
316
286
254
227
202
181
170
160
149
134
113
96
Granzyme
Granzyme
Granzyme
Granzyme
Dad1
Fast K
RVP1
Dr-nm23
Requiem
CAS
Perforin
-L32
GAPDH
A
B
H
3
406
361
316
283
253
227
202
181
163
145
133
-113
96
www.bdbiosciences.com
Product Listing
Template
Protected
hAPO-5
Template
Protected
hAPO-5b
Template
Protected
hAPO-5c
XIAP
TRAF1
TRAF2
CART
NAIP
MIHC
MIHB
TRPM-2
CRAF
--L32
GAPDH
406
361
322
286
253
203
179
163
148
--113
96
TRAF1
TRAF2
CART
I-TRAF
TRAF5
TRAF6
CRAF
TRIP
---L32
GAPDH
361
322
286
253
226
163
148
136
---113
96
XIAP
survivin
NAIP
c-IAP-2
c-IAP-1
TRPM-2
-----L32
GAPDH
406
361
253
203
179
163
-----113
96
Template
Protected
Template
Protected
Template
Protected
hAPO-6
IPL
ASK1
Harakiri
SIAH
DFF
Nip2
Nip3
Nip1
DAP-K
DAP-K
DRM
L32
GAPDH
Template
Protected
mAPO-3
FLICE
FasL
Fas
FADD
FAP
FAF
TRAIL
TNFRp55L
TRADD
RIP
--L32
GAPDH
mAPO-2
mAPO-1
407
361
316
283
256
226
202
181
163
145
133
113
96
FLICE
YAMA
Mch2
Caspase 11
Caspase 12
ICH L/S
Mch3
ICE
Caspase X
caspase-2 (S)
--L32 112
GAPDH
406
325
283
253
226
202
181
163
151
137
---
Template
Protected
97
bclw
bclxL
bclxS
bfl1
bak
bax
bcl2
----bad
L32
GAPDH
406
364
272
239
202
182
160
----139
112
97
rAPO-1
406
353
316
283
226
202
181
160
149
134
--112
97
Fas Antigen
bcl-x(L)
bcl-x(S)
FasL
Caspase-1
Caspase-3
Caspase-2(L)
bax
bcl-2
Caspase-2(S)
--L32
GADPH
406
363(L)
325(S)
286
253
226
202(L)
181
160
137(S)
--113
97
www.bdbiosciences.com
www.bdbiosciences.com
High-quality, affordable
expression profiling
BD Biosciences Clontech offers a
complete platform for the analysis of
differential gene expression that
extends from RNA isolation to
bioinformatics.
Atlas Arrays, the central tool for
expression profiling, consist of
cDNA fragments or long oligos from
hundreds of genes immobilized on a
nylon membrane, plastic film, or
glass slide. Each gene included on an
Atlas Array is well characterized. By
focusing on known genes, Atlas
Arrays provide informative data
immediately. Although Atlas Arrays
provide sophisticated information,
the procedure for using them is
straightforward (Figure 1). The first
step is to prepare cDNA probes from
total or poly A+ RNA. You separately
hybridize probes to the Atlas Array,
perform a high-stringency wash, and
analyze the hybridization pattern.
Labeling and hybridization reagents
are provided.
The relative expression levels of each
gene can be assessed by comparing
the signals obtained from each array.
To automatically analyze Atlas
hybridization
results,
use
AtlasImage 2.01, a comprehensive
data analysis software package. For
visualizing expression patterns
across
multiple
samples,
AtlasNavigator provides a data
visualization and analysis tool to
help you focus on relevant genes. For
the highest quality radioactive
probes, we recommend using the
AtlasPure RNA Labeling System.
When using fluorescent probes, we
recommend using the Atlas Glass
Fluorescent Labeling Kit for uniform
label distribution.
Glass Microarrays
TM
TM
Atlas Glass
Fluorescent Labeling Kit (#K1037-1)
Atlas SMART
Probe Amplification Kit (#K1034-1)
Generate probes
TM
TM
Atlas Plastic
or Nylon Arrays
Atlas Glass
Microarrays
TM
AtlasImage
Fluorescent scanning
software
(#V1211-1)
AtlasNavigator
(#V1220-1)
Obtain Atlas
gene information
TM
AtlasInfo
Profile expression
across tissues
Tissue expression profiling using
TM
Figure 1. Atlas Array products take you from RNA isolation to localization of genes expression.
www.bdbiosciences.com
# of Genes
Targets
Label/detection method
Relative sensitivity
Relative resolution
Imaging equipment
Multiple use
Analysis/ease of use
Yes
Easiest
No membrane deformation
++++
100% tested oligos
Yes
Glass
3,800
Long oligos
33
P
Fluorescence
+++
++
+++
++++
Phosphorimager
Fluorescent
scanner
No
Easiest
No slide deformation
++++
100% tested oligos
Coming soon
Expression profiling
Oligo
array
Hybridization efficiency
cDNA
array
Efficiency
Mutation
detection
0 10 20
100
200
600
1000
Oligos
cDNAs
Figure 2. The approximate length at which a DNA target achieves the optimal hybridization efficiency (yellow) and homologous gene discrimination (red).
www.bdbiosciences.com
Nylon
1,176
PCR-generated cDNA fragments
32
33
P
P
++++
+++
+
++
Phosphorimager or
autoradiogram
Yes
More difficult
Due to membrane deformation
++
100% sequence-verified
No
We deliver:
Phosphorimages of hybridized
membranes
AtlasImage software analysis output, indicating differential gene
expression
Follow-up technical support
For limited sample material we offer
Custom Atlas SMART Probe
Amplification.
10
www.bdbiosciences.com
with the
Baboon hybridization,
Cyno
Rhesus such
Pig as Sheep
Price ($)differences
Table I: Cell types and conditions for the Apoptosis cDNA Panels
HeLafor general apoptosis studies
Control (untreated)
www.bdbiosciences.com
11
Quickly
across a
Body screen
Header
range of apoptotic
Body Sub-header
conditions
G3PDH
1
p21
1
Figure 1. Confirmation of up- or downregulated genes under different apoptotic conditions using the HeLa panel.
Panel A. G3PDH, a housekeeping gene.
Panel B. For the p21 gene, significant
up regulation was seen with 20-hr desferrioxamine treatment (lane 5), the
harshest of the conditions on the panels. Lane 1: control. Lane 2: TNF for 2
hr. Lane 3: TNF for 5 hr. Lane 4: desferrioxamine for 6 hr. Lane 5: desferrioxamine for 20 hr.
Description
Size
Cat. No.
10 rxns
10 rxns
K1440-1
K1441-1
References
1. Siebert PD, Huang BC. 1997: Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines. Proc. Natl.Acad. Sci. USA 94(6):21982203.
2. Human Multiple Tissue cDNA Panels (July 1997) CLONTECHniques XII(3):57.
3. Ossovskaya V.S, Mazo IA, Chernov MV, Chernova OB, Strezoska Z, Kondratov R, Stark GR, Chumakov PM, Gudkov AV.
1996. Use of genetic suppressor elements to dissect distinct biological effects of separate p53 domain. Proc. Natl. Acad. Sci.
USA 93(19):1030910314.
4. Bacus SS, Yarden Y, Oren M, Chin DM, Lyass L, Zellnick CR, Kazarov A, Toyofuku W, Gray-Bablin J, Beerli RR, Hynes NE,
Nikiforov M, Haffner R, Gudkov A, Keyomarsi K. 1996. Neu differentiation factor (Heregulin) activates a p53-dependent
pathway in cancer cells. Oncogene 12(12):25352547.
12
www.bdbiosciences.com
BD Clontech
G ra nzy m e B
TRAIL
TRAIL
FasL
TNF
DR3
Fas
TNFR1
DR
ID
DAXX
FAD
8
s p ase
p ase 2
Ca
L ICE
Cas
p a s e 10
I- F
www.bdbiosciences.com
13
DD
FA P
ase 8
C a s p a s e 10
MA
FAD
C asp
TR A DD
IP
FA F
C as
Gr
an
zy m
eB
DR4
Per forin
Perforin
Untreated
100
B
101
102
103
104
TRAIL
TRADD
FADD
04
100
04
100
101
102
103
104
101
102
103
104
RIP
RIP (receptor interacting protein) is a
serine/threonine kinase which may
be recruited to TNFR type I8 and
Fas9 receptor signal complexes in
activated cells. RIP may interact with
other signal proteins within these
complexes (e.g., RAIDD) and has
also been shown to interact with
pro-caspase-2.
References
1. Griffith, T.S. and T.A. Ferguson. 1997. The role of FasL-induced apoptosis in immune privilege. Immunol. Today 18:240244.
2. Lynch, D.H., F. Ramsdell and M.R. Alderson. 1995. Fas and FasL in the homeosta-tic regulation of immune responses.
Immunol. Today 16: 569-574.
3. Wajant, H., F. Johannes, E. Haas, K.Siemienski, R. Schwenzer, G. Schubert, T. Weiss, M. Grell and P. Scheurich.1998.
Dominant-negative FADD inhibits TNFR60, Fas/Apo1 and TRAIL-R/Apo2-mediated cell death but not gene induction. Curr. Biol.
8:113-116.
4. Yeh, W.C., J.L. Pompa, M.E. McCurrach, H.B. Shu, A.J.Elia, S. Shahinian, M. Ng., A. Wakeham, W. Khoo, K. Mitchell, W.S.
El-Diery, S.W. Lowe, D.V. Goeddel and T.W. Mak. 1998. FADD: essential for embryo development and signaling from some,
but not all, inducers of apoptosis. Science 279:1954-1958.
5. Muzio, M., A.M. Chinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Bretz, M. Zhang, R. Gentz,
M. Mann, P.H. Krammer, M.E. Peter and V.M. Dixit. 1996. FLICE, a novel FADD-homologue ICE/CED-3-like protease, is
recruited to the CD95 (Fas/APO-1) death-inducing signaling complex. Cell 85: 817-827.
6. Hsu, H., J. Xiong and D.V. Goeddel. 1995. The TNF receptor-1 associated protein TRADD signals cell death and NF- B activation. Cell 81:495-504.
7. Varfolomeev, E.E., M.P. Boldin., T.M. Goncharov and D. Wallach. 1996. A potential mechanism of cross-talk between the
p55 tumor necrosis factor receptor and Fas/Apo-1: proteins binding to the death domains of the two receptors also bind to
each other. J. Exp. Med. 183:1271-1275.
8. Ting, A.T., F.X. Pimentel-Muinos and B. Seed.1996. RIP mediates tumor necrosis factor receptor 1 activation of NF- B but
not Fas/APO-1-initiated apoptosis. EMBO J. 15:6189-6196.
9. Stanger, B.Z., P. Leder, T.H. Lee, E. Kim and B. Seed. 1995. RIP: a novel protein containing a death domain that interacts
with Fas/APO-1 (CD95) in yeast and causes cell death. Cell 81:513-523.
14
www.bdbiosciences.com
c-IAP-1
10. Hsu, H., I. Solovyev, A. Colombero, R. Elliott, M. Kelley and W. J. Boyle. 1997. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5. J. Biol. Chem. 272:13471-13474
11. Cheng, G., A.M. Cleary, Z. Ye, D. Hong, S. Lederman and D. Baltimore. 1995. Involvement of CRAF1, a relative of TRAF,
in CD40 signaling. Science 267:1494-1498.
12. Mosialos, G., M. Birkenbach, R. Yalamanchili, T. VanArsdale, C. Ware and E. Kieff. 1995. The Epstein-Barr virus transforming protein LMP1 engages signaling pro-teins for the tumor necrosis factor receptor family. Cell 80:389-399.12.
13. Regnier, C.H., C. Tomasetto, C. Moog-Lutz, M.P. Chenard, C. Wendling, P. Basset and M.C. Rio. 1995. Presence of a new
conserved domain in CART1, a novel member of the tumor necrosis factor receptor-associated protein family, which is
expressed in breast carcinoma. J. Biol. Chem. 270:25715-25721.
14. Aizawa, S., H. Nakano, T. Ishida, R. Horie, M. Nagai, K. Ito, H. Yagita , K. Okumura, J. Inoue and T. Watanabe. 1997. Tumor
necrosis factor receptor-associated factor (TRAF) 5 and TRAF2 are involved in CD30-mediated NFkappaB activation. J. Biol.
Chem. 272:2042-2045.
15. Ishida, T.K., T. Tojo, T. Aoki, N. Kobayashi, T.Ohishi, T. Watanabe, T. Yamamoto and J. Inoue. 1996. TRAF5, a novel tumor
necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc. Natl. Acad. Sci. USA 93:9437-9442.
16. Ishida, T., S. Mizushima, S. Azuma, N. Kobayashi, T. Tojo, K.Suzuki, S. Aizawa, T. Watanabe, G. Mosialos, E. Kieff, T.
Yamamoto and J. Inoue. 1996. Identification of TRAF6, a novel tumor necrosis factor receptor-associated factor protein that
mediates signaling from an amino-terminal domain of the CD40 cytoplasmic region. J. Biol. Chem. 271:28745-28748.
17. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama and D.V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature
383:443-446.
18. Rothe, M., M.B. Pan, W.J. Henzel, T.M. Ayres and D.V. Goeddel. 1995. The TNFR2-TRAF signaling complex contains two
novel proteins related to baculoviral inhibitor of apoptosis proteins. Cell 83:1243-1252.
19. Tsitsikov, E.N., D.A. Wright and R.S. Geha. 1997: CD30 induction of human immunodeficiency virus gene
transcription is mediated by TRAF. Proc. Natl. Acad. Sci. USA 94:1390-1395.
www.bdbiosciences.com
15
kD
200
116
97
DR3
66
55
36
31
21
Western blot analysis of DR3, a receptor for the cytotoxic ligand TRAIL, in
Jurkat T cell lysate. Affinity purified,
rabbit anti-human DR3 (Cat. No.
556566) identifies DR3 as an ~ 59 kD
band.
Product Listing
Description
Clone(s)
Specificity
Daxx
DcR1
DR3
DR4
DR4
DR6
DRAK2
FADD
Fas
Polyclonal
Rabbit
Polyclonal
Polyclonal
Polyclonal
Rabbit
Rabbit
A66-2
DX2
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Fas
Fas
G254-274
Jo2
Hu
Ms
NOK-1
NOK-2
G247-4
Kay-10
Hu
Hu
Hu
Ms
Fas Ligand
Granzyme B
Mcl-1
Perforin
Perforin Antibody Reagent Set
RIP
SODD
TRADD
TNF-
MFL3
2C5/F5
Polyclonal
dG9
dG9,27-35
G322-2
Rabbit
B36-2
MAb11
Ms
TNF-
MP6-XT22
Ms
TNF-
359-81-11
Hu
TRAF3
TRAF3
TRAIL
TRAIL
TRAIL
Polyclonal
B1-6
B35-1
RIK-1
RIK-2
Hu
Hu
Hu
Hu
Hu
Fas
Fas
Fas
Fas
Ligand
Ligand
Ligand
Ligand
Hu
Hu
Hu
Hu
Hu
Hu
Applications
Format
WB
WB
WB
WB
WB
WB
WB
IP, WB
FA, FC
Size
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
NA/LE
Purified
FC
Biotin
FC
FITC
FC
PE
FC
CyChr
FC
APC
WB
Purified
IP, FC, FA
NA/LE
Purified
Biotin
FITC
PE
IP, FC, FA
NA/LE
IP, FA
NA/LE
IP, WB, FC
Purified
FC, FA
NA/LE
FC
Purified
FC
Biotin
FC
PE
FC
Purified
WB
Purified
IP, WB, IHCF, IHCP Serum
WB
Purified
FC
PE Set
IP, WB
Purified
WB
Purified
WB
Purified
FC
Purified
FC
FITC
FC
PE
FC
APC
FC
Purified
FC
FITC
FC
PE
FC
APC
FC, FA
NA/LE
FC
Purified
FC
PE
WB, IHCP
Serum
WB
Purified
WB
Purified
FC
Purified
FC
Purified
FC
Biotin
FC
PE
50 g
0.1 mg
50 g
50 g
50 g
50 g
200ul
0.1 mg
0.5 mg
0.1 mg
100 tests
100 tests
100 tests
100 tests
100 tests
0.1 mg
0.5 mg
0.5 mg
0.5 mg
0.5 mg
0.2 mg
0.25 mg
0.25 mg
0.1 mg
0.5 mg
0.5 mg
0.5 mg
0.2 mg
0.5 mg
50 g
0.1 ml
0.1 mg
100 tests
0.1 mg
200 l
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.5 mg
0.1 mg
0.1 mg
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.2 mg
0.1 mg
67051N
80056E
67061N
66901N
66891N
69481N
80446E
65751A
33450D
33451D
33452X
33454X
33455X
33458X
33459X
65311A
15400D
15401D
15402D
15404D
15405B
65320C
65330C
65431A
09930D
09931D
09932D
09935B
28101D
69901N
13656E
65991A
6599KK
65591A
80586E
66431A
18641A
18644A
18645A
18649A
18131A
18134A
18135A
18139A
18910D
18911A
18915A
66536E
66191A
66251A
69891A
69431A
69432B
69435A
556565
550622
556566
556544
556543
550439
550945
556402
555670
555671
555672
555673
555674
559773
558814
556370
554254
554255
554256
554257
554258
556371
556375
556387
553851
553852
553853
553854
555291
550558
554103
556434
556437
556395
550857
556496
554510
554512
554513
554514
554416
554418
554419
554420
554553
554554
554556
556506
556461
556468
550517
550515
550431
550516
IP, FC, FA
FA
FA
FA
E
E
E
100 g
10 g
10 g
10 g
20 x 96 tests
20 x 96 tests
20 x 96 tests
67231A
19761T
19321T
19771T
2649KI
2637KI
2673KI
556578
554618
554589
554619
555224
555212
555268
Related Reagents
16
www.bdbiosciences.com
Hu
Ms
Hu
Hu
Hu
Ms
Purified
Purified
Purified
Purified
Set
Set
Set
BD Pharmingen
3.2. Caspases
eB
zy m
an
APAF-
C a s p a s e 10
C as
C as
spa
me
p ase 4
p ase 8
C a s p ase 7
C a s p ase 3
C as
C a s p ase 9
p ase 1
C a s p ase 6
CrmA
C a s p ase 2
cI
XIA P
A P1 cIA P
SR EBP
A P -2 4
D 4 - G DI
D FF
L a m in s
PA RP
p17- 37
p17- 20
Pro-domain
p10-12
NH2
COOH
Pro-Caspases
Ca
C y to chro
se 8
Gr
Per forin
Per forin
Per forin
Per forin
Nuclear Collapse
DNA fragmentation
Death
Activation by Cleavage
= Cleavage Site
Active Caspases
Activation of Caspases.
Caspases are synthesized as inactive proenzymes (pro-caspases) that are processed in cells undergoing apoptosis by
self-proteolysis and/or cleavage by another protease. The processed forms consist of large (17-20 kD) and small (1012 kD) subunits which associate to form an active enzyme.
www.bdbiosciences.com
17
WB IP/WB
116
97
66
55
Caspase-7
kD
116
97
kD
66
36
31
p17
14
kD
116
97
66
55
kD
116
97
66
55
p34
36
31
Caspase-6
116
97
66
55
21
14
p11
p32
21
1 2 3 4 5
36
31
15
2
CrmA
14
Caspase-2
18
1
21
29
36
31
p33
44
36
31
Caspase-3
Caspase-1
67
Caspase-8
21
55
p45
kD
kD
200
6
1
CrmA
Western blot analysis of caspase-2.
Jurkat T cell lysates (lane 1), mouse myeloblasts (lane 2)
and DC-3 SV40-transformed, rat ovarian granulosa cells
(lane 3) were probed with anti-caspase-2 (clone G3101248, Cat. No. 554131). The G310-1248 antibody identifies caspase-2 (long form) as an ~48 kD band.
36
31
21
1
References
1. Cerretti, D.P., C.J. Kozlosky, B. Mosley, N. Nelson, K. Van Ness, T.A. Greenstreet, C.J. March, S.R. Kronheim, T. Druck, L.A. Cannizzaro, K. Heubner and R.A. Black. 1992. Molecular cloning
of the interleukin-1 converting enzyme. Science 256:97-100.
2. Komiyama, T., L.T. Quan and G.S. Salvesen. 1996. Inhibition of cysteine and serine proteinases by the cowpox virus serpin CrmA. Adv. Exper. Med. Biol. 389:173-176.
3. Tewari, M., L.T. Quan, K. ORourke, S. Desnoyers, Z. Zeng., D.R. Beidler, G.G. Poirier, G.S. Salvesen and V.M. Dixit. 1995. Yama/CPP32, a mammalian homolog of CED-3 is a CrmAinhibitable protease that cleaves the death substrate poly (ADP ribose) polymerase. Cell 81:801-809.
4. Zhou, Q., S. Snipas, K. Orth, M. Muzio, V.M. Vixit and G.S. Salvesen. 1997. Target protease specificity of the viral serpin CrmA. J. Biol. Chem. 272:7797-7800.
18
www.bdbiosciences.com
Product Listing
Description
Clone(s)
Specificity
Apaf-1
Apaf-1
Caspase-3, Active
Caspase-1
Caspase-10
Caspase-14
Caspase-14
Caspase-14
Caspase-2
Caspase-3
Caspase-3
Caspase-3
Caspase-3, Active
Caspase-3, Active
Caspase-3, Active
Caspase-3,
Active /FITC Mab Apoptosis Kit
Caspase-3,
Active Active- ELISA Ab Pair
Caspase-3, Active- ELISA Set
Active/PE MAb Apoptosis Kit
Caspase-3,
Caspase-4
Caspase-6
Caspase-7
Caspase-8
Caspase-8
Caspase-9
Caspase-9
Caspase-9
Caspase-9
c-IAP
Crm A
Cytochrome c
Cytochrome c
Hsp60
Hsp60
I-FLICE
Nedd4
Nedd4
Nedd4
24
24
C92-605
B24-2
Polyclonal
70A1426
32
32
G310-1248
Polyclonal
Polyclonal
Polyclonal
Polyclonal
C92-605
C92-605
C92-605
BD Pharmingen
BD Transduction Laboratories
Applications
Format
Size
Hu
Hu
Hu,Ms
Hu,Ms,R
Hu
Hu, Ms
Hu, Ms
Hu, Ms
Hu, Ms
Hu, Ms
Hu,Ms
Hu,Ms
Hu,Ms
Hu,Ms
Hu,Ms
Hu,Ms
WB, IF
WB, IF
FC, IF, IP
WB
WB
WB
WB, IF
WB, IF
WB
WB, IHCP
IHCF
FC
IP,IHCF,FC
FC
FC
FC
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Biotin
PE
Purified
Biotin
PE
Kit
50 g
150 g
25 g
0.1 mg
50 g
150 g
50 g
150 g
0.1 mg
0.1 ml
0.1 mg
100 tests
0.1 mg
100 tests
100 tests
100 tests
A92820
68651G
68651G
66441A
67041N
80641F
C97020-50
C97020-150
13951A
65906E
67342A
67345X
67341A
68652X
68655X
6976KK
611364
611365
559565
556497
556564
550873
611510
611511
554131
556425
557038
557091
557035
550557
550821
550480
19 & C92-605
ELISA
ELISA
Pair
0,5 ml ea.
69941K
550578
19 & C92-605
ELISA
ELISA
Set
Set
8072KK
550930
C92-605
B25-1
B93-4
B94-1
B9-2
Rabbit
B40
Polyclonal
Rabbit
Rabbit
B75-1
A71-1
6H2.B4
7H8.2C12
24
24
Polyclonal
Rabbit
15
15
Hu, Ms
Hu,Ms,R
Hu
Hu
Hu
Hu, Ms
Hu
Hu
Hu
Hu
Hu
Viral Protein
Hu, Ms, R
Hu, Ms, R
Hu, Ms, R
Hu, Ms, R
Hu
Hu, Ms
Ms
Ms
FC
WB
WB
IP, WB
WB
WB
WB
WB
WB
WB
IP, WB
WB
IP
WB
WB
WB
WB
WB
WB, IF
WB, IF
Kit
Purified
Purified
Purified
Purified
Serum
Purified
Serum
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Purified
Purified
Kit
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0,1 ml
0.1 mg
0.1 ml
50 g
50 g
0.1 mg
0.1 mg
0.1 mg
0.1 mg
150 g
150 g
50 g
0,1 ml
50 g
150 g
8067KK
66171A
68041A
66871A
66231A
69236E
66571A
68086E
69461N
69471N
66791A
65921A
65971A
65981A
H99020-050
H99020-150
67071N
69976E
N95520-050
N95520-150
550914
556459
556581
556541
556466
559932
556510
556585
550437
550438
556533
556427
556432
556433
611562
611563
556567
550598
611480
611481
www.bdbiosciences.com
19
20
www.bdbiosciences.com
D4-GDI
* Na, S., et al. 1996. D4-GDI, a substrate of CPP32, is proteolyzed during Fas-induced apoptosis.
J. Biol. Chem. 271:11209-11213.
DFF
* Liu, X., et al. 1997. DFF, a heterodimeric protein that
functions downstream of caspase-3 to trigger DNA
fragmentation during apoptosis. Cell 89:175-184.
DNA-dependent protein kinase
* Casciola-Rosen, L., et al. 1996. Apopain/CPP32 cleaves
proteins that are essential for cellular repair: A fundamental principle of apoptotic cell death. J. Exp. Med.
183:1957-1964.
* Song, Q., et al. 1996. DNA-dependent protein kinase
catalytic subunit: A target for an ICE-like protease in
apoptosis. EMBO J. 15:3238-3246.
MEKK
* Cardone, M.H. et al. 1997. The regulation of anoikis:
MEKK-1 activation requires cleavage by caspases. Cell 90:
315-323.
MDM2
* Nicholson, D.W., et al. 1997. Caspases: killer proteases.
Trends Biochem. Sci. 22:299-306.
* Erhardt, P., et al.1997. Identification of the MDM2
oncoprotein as a substrate for CPP32-like apoptotic
proteases. J. Biol. Chem. 272:15049-15052.
PAK1/PAK2
* Walter, B.N., et al. 1998. Cleavage and activation of
p21-activated protein kinase gamma-PAK by CPP32
(caspase 3). Effects of autophosphorylation on activity. J.
Biol. Chem. 273:28733-28739.
PARP
* Nicholson, D.W., et al. Identification and inhibition of
the ICE/CED-3 protease necessary for mammalian
apoptosis. Nature 376:37-43.
* Tewari, M., et al. 1995. Yama/Cpp32b, a mammalian
homologue of CED-3, is a CrmA inhibitable protease
that cleaves the death substrate poly (ADP-ribose) polymerase. Cell 81:801-809.
SREBP-1,2
* Emoto, Y., et al. 1996. Cleavage of sterol regulatory element binding proteins (SREBPs) by CPP32 during
apoptosis. EMBO J. 15:1012-1020.
Rb
* Tan, X., et al. 1997. Degradation of retinoblastoma protein in tumor necrosis factor- and CD95-induced cell
death. J. Biol. Chem. 15:9613-9616.
For a more complete review of cellular proteins which are
targets of caspase-mediated cleavage, please see
Tan, X., et al. 1998. The caspase-RB connection in cell
death. Trends Cell Biol. 8:116-120.
Control
Staurosporine
Immunofluorescence Micrographs of
Cleaved PARP. In this experment, HeLa cells
were induced to undergo apoptosis with
staurosporine. The fluorescent micrographs
shown indicate that cleavage site-specific
PARP-FITC antibodies (polyclonal, Cat. No.
551528) stain the staurosporine-treated (B),
but not the control cells (A). A ' and B '
represent phase contrast correlates of A and
B respectively.
Product Listing
Description
Clone(s)
Specificity
D4-GDI
D4 GDI
Rho-GDI/D4-GDI
DFF (C-terminal)
DFF (C-terminal) Blocking Peptide
DFF (N-terminal)
DFF (N-terminal) Blocking Peptide
DNA-PK (p350)
DNA-PK (p350)
IL-1
PAK1/PAK2
PARP
PARP
PARP
PKC
Retinoblastoma Protein (Rb)
SREBP-1
SREBP-2
Polyclonal
97A1015
Polyclonal
Polyclonal
Hu
Hu, Ms
Hu
Hu
Polyclonal
Hu
Polyclonal
4F10C5
B122
Polyclonal
7D3-6
4C10-5
C2-10
MC5
G3-245
IgG-2A4
IgG-1C6
Hu,Ms,R
Hu,Ms,R
Ms
Hu
Hu,Ms,R
Hu
Hu,Ms
Hu,Ms,R
Hu,Ms,R
Hu
Hu
Applications
Format
Size
WB
WB
WB
WB
EBS
WB
EBS
IP,WB
IP,WB
FA
WB
IP,WB,FC
IP,WB,FC
WB
IP,WB,IHC
IP,WB,IHC
WB
WB
Serum
Purified
Serum
Purified
Peptide
Purified
Peptide
Serum
Purified
Purified
Serum
Purified
Purified
Ascites
Purified
Purified
Purified
Purified
0.1 ml
50 g
0.1 ml
50 g
0.1 mg
50 g
0.1 mg
0.1 ml
0.1 mg
0.25 mg
0.1 ml
0.1 mg
0.1 mg
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.1 mg
66456E
80521N
66586E
66921N
67281A
66911N
67291A
65586E
66141A
23410C
67416E
66391A
66401A
65196E
14871A
14001A
67351A
67361A
556498
550746
556511
556546
556995
556545
556996
556394
556456
557539
557053
556493
556494
556362
554207
554136
557036
557037
Western blot analysis of Caspase-3, D4-GDI and PARP during Fas-induced apoptosis. Jurkat
T cells were treated with anti-human Fas mAb, clone DX2 (Cat. No. 555670) and Protein G.
Cultures were harvested at various time points as indicated and analyzed by western blotting
using antibodies that recognize both the intact and cleaved forms of caspase-3 (polyclonal, Cat.
No. 556425), D4-GDI (polyclonal, Cat. No. 556511) and PARP (clone 4C10-5, Cat. No. 556494).
The intact forms of the proteins were detected at all time points; however, the cleaved forms
(caspase-3 [17 kD], D4-GDI [23 kD] and PARP [85 kD]) were only detected at 2 hr and beyond
after the induction of apoptosis.
BD Pharmingen
www.bdbiosciences.com
21
10 0
10 1
10 2
10 3
10 4
Fluorescence Intensity
Flow cytometric analysis of Bcl-2 in human
peripheral blood lymphocytes. Cells were stained
with Bcl-2/100 Antibody Reagent Set (Cat. No.
556357), which includes anti-human Bcl-2-FITC
(clone Bcl-2/100) and an IgG 1 -FITC isotype control.
kD
66
55
36
31
Bax
21
14
6
1
kD
200
116
97
66
55
36
31
Bcl-X
21
1
22
www.bdbiosciences.com
-3
4-3
f-1
Ra
Cytochro
bag
bad
bad
Cytochrome C
ba d
bcl-XL
bcl-XL
bax
AIF
Raf-1
ba d
bag
bcl-2
bcl-2
B I-1
bcl-2
bid
bcl-2
bax
bid
bax
bax
bax
bid
me
Product Listing
Description
Clone(s)
Specificity
Bad
Bad
Bak
Bak
Bak
Bax
Bax
Bax
Bax
Bax
Bid
Bid
Bid
Bcl-2
Bcl-2
Bcl-2 Antibody Reagent Set
P3F6
2G11
Polyclonal
G317-1
G317-2
6A7
G206-1276
Polyclonal
Polyclonal
4D2
Rabbit
7
7
Polyclonal
Bcl-2/100
Bcl-2/100, MOPC-21
Hu
Ms
Hu
Hu
Hu
Hu,
Hu,
Hu
Ms,
Ms
Hu,
Hu
Hu
Hu
Hu
Hu
Bcl-2
Bcl-2
Bcl-2 Antibody Reagent Set
4D7
6C8
6C8, Ha4/8
Hu
Hu
Hu
Bcl-2
Bcl-2 Antibody Reagent Set
Polyclonal
3F11, A19-3
Ms
Ms
Bcl-2
Bcl-w
Bcl-X
Bcl-X
Boo
Nip-1
Nip-1
Polyclonal
16H12
Polyclonal
2H12
D12-1932
5
5
Ms,
Hu,
Hu,
Hu,
Ms
Hu,
Hu,
Ms, R
Ms
R
Ms
R
Ms
Ms
Ms, R
R
R
Format
Applications
Size
Purified
Purified
Serum
Purified
Purified
Purified
Purified
Serum
Serum
Purified
Serum
Purified
Purified
Serum
Purified
FITC Set
PE Set
Purified
Purified
FITC Set
PE Set
Serum
FITC Set
PE Set
Serum
Purified
Serum
Purified
Purified
Purified
Purified
WB, IHC(P)
IP, WB
WB
WB, IHC(F)
WB
IP, WB
IP, WB, IHC(F), IHC(P)
IP, WB, IHC(F), IHC(P)
IP, WB, IHC(F), IHC(P)
IP
WB, IP
WB, IF
WB, IF
IP, WB, FC, IHC(F), IHC(P)
WB
FC
FC
IP, WB
IP, WB, FC, IHC(F)
FC
FC
IP, WB, IHC(F), IHC(P)
FC
FC
IP, WB, IHC(F), IHC(P)
WB
WB, IHC(F), IHC(P)
WB, IHC(P)
WB
WB, IF
WB, IF
0.1 mg
0.1 mg
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 ml
0.1 ml
0.1 mg
0.1 ml
50 g
150 g
0.1 ml
0.1 mg
100 tests
100 tests
0.1 mg
0.1 mg
100 tests
100 tests
0.1 ml
100 tests
100 tests
0.1 ml
50 g
0.1 ml
0.1 mg
50 g
50 g
150 g
66551A
13361A
66026E
65401A
65371A
66241A
13401A
13666E
13686E
13421A
69356E
697920-050
B97920-150
14371E
65111A
6511KK
6681KK
14831A
15131A
1513KK
6682KK
15616E
1502KK
6683KK
13456E
69911N
65186E
66461A
80611N
N79420-050
N79420-150
556508
554078
556440
556384
556382
556467
554082
554104
554106
554084
550365
611528
611529
554160
556354
556357
556535
554202
554231
554234
556536
554279
554221
556537
554087
550559
556361
556499
550860
611096
611097
EBS
EBS
Peptide Set
Peptide Set
0.1 mg ea
0.1 mg ea
67171K
68001K
556573
556579
Related Reagents
BD Pharmingen
BD Transduction Laboratories
www.bdbiosciences.com
23
3.4. Cytochrome c
References
1. Boyer, P.D., B. Chance, L. Ernster, P. Mitchell, E. Racker and E.C.Slater. 1997. Oxidative phosphorylation and photophosphorylation. Annu. Rev. Biochem. 46:955-1026.
2. Liu, X., C.N. Kim, J. Yang, R. Jemmerson and X. Wang.1996. Induction of apop-totic program in cell-free extracts:
requirement for dATP and cytochrome c. Cell 86:147-157.
3. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X. 1997. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90(3):405-13.
4. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91(4):479-89.
5. Duckett, C.S., F. Li, Y. Wang, K.J. Tomaselli, Thompson, C.B. and R.C. Armstrong.1998. Human IAP-like protein regulates
programmed cell death downstream of Bcl-X L and cytochrome C. Mol. Cell Biol. 18:608-615.
6. Rosse, T., O. Reynald, L. Monney, M. Rager, S. Conus, I. Fellay, B. Jansen and C. Borner. 1988. Bcl-2 prolongs cell survival
after Bax-induced release of cytochrome c. Nature 391:496-499.
7. Yoshida, H., K. Young-Yun, R. Yoshida, A.J. Elia, A. Hakem, R. Hakem, J.M. Pen-ninger and T.W. Mak. 1998. Apaf1 is
required for mitochondrial pathways of apoptosis and brain development. Cell 94:739-750.
24
www.bdbiosciences.com
Product Listing
Description
Clone(s)
APAF-1
Apaf-1
Apaf-1
Caspase-9
Caspase-9
Caspase-9
Caspase-9
Cytochrome c
Cytochrome c
Polyclonal
24
24
B40
Polyclonal
Rabbit
Rabbit
6H2.B4
7H8.2C12
Specificity
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu, Ms, R
Hu, Ms, R
Applications
Format
Size
WB
WB, IF
WB, IF
WB
WB
WB
WB
IP
WB
Serum
Purified
Purified
Purified
Serum
Purified
Purified
Purified
Purified
0.1 ml
50 g
150 g
0.1 mg
0.1 ml
50 g
50 g
0.1 mg
0.1 mg
kD
kD
200
66
55
116
97
66
55
36
31
21
14
6
Cytochrome c
1
BD Pharmingen
BD Transduction Laboratories
556584
611364
611365
556510
556585
550437
550438
556432
556433
36
31
21
Cytochrome c
14
6
1
www.bdbiosciences.com
25
36
31
21
1 2
Pa
M
Western blot analysis of JNKK in HeLa human carcinoma cell lysates. Lane 1, clone A32-1 (Cat. No
556400). Lane 2, mouse IgG 1 isotype control.
Clone A32-1 identifies JNKK as an ~45 kD band.
C as
p ase 3
JN
ac1
NIK
Growth
Factor
KKs
SK
20
1
as
K3
38
NF
ct k
iv e
K6
kD
200
Ik B
FkB
JN
Ik
k1
Ikk2 Ikk3
EKK
af
From
TACI
EK
RK
116
97
KK
Cytoplasm
Jun
66
55
36
31
Cell
Activation
21
1
Western blot analysis of IKK in Daudi B lymphoma cell lysate. Lane 1, anti-IKK , clone B78-1
(Cat. No 556532). Lane 2, a mouse lgG 2b isotype
control. Clone B78-1 identifies IKK as an ~85 kD
band.
kD
200
116
97
NIK
66
55
36
31
21
1
26
www.bdbiosciences.com
Product Listing
Description
Clone(s)
Specificity
A20
ASK1
c-myc
IB
IB
IB
IKK
IKK
IKK
IKKB/IKK2
IKK/NEMO
IKK/NEMO
JNK1
JNK1
JNKK
JNKK
LITAF
LITAF
MEK1
MEK2
MEK2
MEKK1
NIK
N-myc
N-myc
PKR
PKR
PTEN
PTEN
E5-1619
Polyclonal
9E10
6A920
21
21
B78-1
Rabbit
Rabbit
10A9B6
54
54
G151-333
G151-666
G282-114
A32-1
30
30
Polyclonal
A7-1
Polyclonal
Polyclonal
Polyclonal
B8.4.B
N-Myc-2
23
23
2
2
Hu
Hu
Hu
BD Pharmingen
BD Transduction Laboratories
Hu,
Hu,
Hu
Hu
Hu
Hu,
Hu,
Hu,
Hu
Hu
Hu
Hu
Hu
Hu
Hu,
Hu,
Hu,
Hu,
Hu
Hu
Hu,
R
R
Hu,
Hu,
Ms, R
Ms, R
Ms, R
D, R
D, R
Ms
Ms
Ms
Ms
Ms
Ms, R
Ms, R
Applications
Format
Size
WB
WB
WB, IHC(P)
WB
WB, IF
WB, IF
IP/WB
WB
WB
WB
WB, IF
WB, IF
IP, WB, IVK
IP, WB
IP, WB
IP, WB
WB
WB
WB
WB
WB
WB
IP/WB
IP/WB
IP, WB
WB, IF
WB, IF
WB, IF
WB, IF
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Purified
Serum
Serum
Purified
Purified
Purified
Purified
Purified
Purified
Purified
50 g
50 g
0.1mg
50 g
50 g
150 g
0.1 mg
50 g
50 g
50 g
50 g
150 g
0.1 mg
0.1 mg
0.1 mg
0.1 mg
50 g
150 g
0.1 ml
0.1 mg
0.1 ml
0.1 ml
50 g
0.1 mg
0.1 mg
50 g
150 g
50 g
150 g
550859
556580
554205
550620
611408
611409
556532
550444
550445
550621
611306
611307
554286
554285
554105
556400
611614
611615
554097
556386
554098
556542
556569
556438
554206
611514
611515
611500
611501
www.bdbiosciences.com
27
10 0
10 1
10 2
10 3
10 4
10 0
10 0
10 1
10 2
10 3
10 4
10 1
10
10 3
10 4
10 1
10
10 3
10 4
10 0
Fluorescence Intensity
28
www.bdbiosciences.com
Product Listing
Description
Clone(s)
Specificity
Applications
Format
Size
p33ING
p53
p53
p53
Polyclonal
G59-12
PAb 122
PAb 240
Hu,
Hu,
Hu,
Hu,
p53
p53
p53 Antibody Reagent Set
DO-1
DO-7
DO-7,27-35
Hu
Hu
Hu
WB, IF
IP, WB, IHC(F), IHC(P)
IP, WB, FC
IP, WB, IHC(F)
IP, WB,IHC(F)
IP, WB, IHC(F), IHC(P)
IP, WB, FC, IHC(F), IHC(P)
FC
p53
PAb 1801
Hu
p53
Rb Antibody Reagent Set
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
-Amyloid (a.a. 1-40)
-Amyloid (a.a. 1-42)
-Amyloid (a.a. 1-43)
PAb 246
Ms
G3-245, MOPC-21 Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
Hu
IP
FC
FC
IP, WB, IHC(F)
IP, WB, IHC(F)
IP, WB, IHC(F)
WB
IP, WB
IP, WB
IP, WB
IP, WB, IF
IP
IP
E, IHC(F)
E, IHC(F)
E, IHC(F)
Serum
Purified
Purified
Purified
Purified
Purified
Purified
FITC Set
Peptide Set
Purified
Purified
Purified
FITC Set
Peptide Set
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Purified
Serum
Serum
Serum
0.1 ml
0.1 mg
0.1 mg
0.1 mg
0.25 mg
0.1 mg
0.1 mg
100 tests
100 tests
0.1 mg
0.25 mg
0.1 mg
100 tests
100 tests
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
0.1 mg
25 g
25 g
25 g
66156E
14211A
14091A
14461A
14461C
15791A
15801A
1580KK
6680KK
14471A
14471C
14451A
6684KK
6685KK
14021A
14011A
14031A
14421A
14441A
14411A
14051A
14061A
14041A
14071A
66471G
66481G
66491G
556457
554157
554147
554166
554167
554293
554294
554298
556534
554169
554170
554165
556538
556539
554141
554140
554142
554163
554164
554162
554144
554145
554143
554146
556500
556501
556502
Hu
Hu
Hu
EBS
EBS
EBS
EBS
EBS
EBS
Peptide Set
Peptide Set
Peptide Set
Peptide
Peptide
Peptide
0.1 mg ea
0.1 mg ea
0.1 mg ea
250 g
250 g
250 g
67111K
67141K
66671K
66501C
66511C
66521C
556571
556572
556521
556503
556504
556505
G4-340
G3-349
C36
G99-73
G99-549
G99-2005
XZ55
XZ91
XZ104
XZ133
Polyclonal
Polyclonal
Polyclonal
Ms
Ms, R
Ms, R
Ms, R
Related Reagents
BD Pharmingen
www.bdbiosciences.com
29
Jurkat
Jurkat
Jurkat
Jurkat
Jurkat
+
+
+
+
+
Staurosporine
Staurosporine
Staurosporine
Staurosporine
Staurosporine
Applications
Format
Size
Cat. No.
WB
WB
WB
WB
WB
Lysate
Lysate
Lysate
Lysate
Lysate
500 g
500 g
500 g
500 g
50 g each
611627
611628
611629
611630
611626
611627
611628
611629
611630
611626
Description
Applications
Format
Size
Cat. No.
WB
WB
WB
WB
WB
IP
WB
WB
Purified
Purified
Purified
Purified
Purified
Purified
Lysate
Purified
100 g
100 g
100 g
100 g
10 g
100 g
0.5 ml
10 g
67281A
67291A
67261A
67271A
16341T
67231A
16226Y
66011T
556995
556996
556993
556994
554336
556578
554327
556439
30
www.bdbiosciences.com
BD Pharmingen
BD Transduction Laboratories
Apoptosis I
0h 0.5h 1h 4h 8h
0h 0.5h 1h 4h 8h
Screening
Analyze your experimental conditions with an array of antibodies to
identify which proteins to target for
further investigation.
Convenient
Each antibody is supplied in a 10 g
quantity and is ready for use.
Economical
Why pay much more to purchase
each antibody separately? Our
Antibody Sampler Kits allow you to
examine multiple proteins of interest
with minimal cost.
Format
Cat. No.
Kit
611427
Protein
Cat. No.
Apaf-1
Bad
Bax
Bcl-2
Bcl-x
BRUCE
CAS
hILP
Mcl-1
Nip1
p53
A92820
130
+
B36420
23kDa +
B73520
21kDa +
B46620
26kDa + nat/den
B61220
26kDa +
B84220 528kDa +
C42920 100kDa + nat/den
H59520 57kDa +
M54020 42/40kDa +
N79420 26kDa +
P21020
53kDa + nat/den
MW
WB
IP
IF IH
+
+
+
+
+
+
+
+
+
+
+
Apoptosis II
Reliable
All BD Transduction Laboratories
antibodies are developed in-house
and subjected to extensive screening
and quality control.
Controls
In addition to antibodies, each kit
contains the appropriate positive
control lysates for Western blotting at no additional charge!
0h 0.5h 1h 4h 8h
PARP
RIP
Caspase-2
Fas
DFF45
Fas Ligand
Caspase-7
TRADD
mCaspase-3
hCaspase-3
FADD
Comprehensive
Each antibody kit targets both established and novel proteins within specific areas of research.
BRUCE
Apaf-1
CAS
hILP
p53
Mcl-1
Nip1
Bcl-x
Bcl-2
Bad
Bax
BD Transduction Laboratories
Antibody Sampler Kits provide a
unique way to examine your samples
for multiple proteins of interest without the costs of purchasing individual antibodies from a variety of
sources. Each kit is built around a
particular area of research and contains multiple antibodies. Since our
monoclonal antibodies are of the
highest quality and specificity, each
produces a distinct signal for its target protein. Each kit also includes
the appropriate positive control
lysates for Western blotting so you
can be assured of your results.
Format
Cat. No.
Kit
611428
Protein
Cat. No.
Caspase-2
Caspase-3
Caspase-3
Caspase-7
DFF45
FADD
Fas/CD95
Fas Ligand
PARP
RIP
TRADD
I75620
48kDa +
+
C31720
32kDa + nat/den +
C76920
32kDa +
M64620 35kDa +
D76320 45kDa +
F36620
24kDa +
+
F22120
45kDa +
+
F37720
37kDa +
den
+
P76420 113kDa +
+
R41220
74kDa + nat/den +
T50320
34kDa +
den
+
MW
WB
IP
IF IH
hILP
Caspase-7
RIP
{
DFF45
Nip 1
Caspase-2
Fig. 1 Jurkat cells were exposed to 4 M staurosporine for up to 8 hrs to induce apoptosis. A
variety of antibodies from our Apoptosis I and II Sampler Kits were used to probe Western blots
of apoptotic Jurkat cells.
www.bdbiosciences.com
31
Features
A unique advancement in proteomics that measures changes in
protein expression
Complementary to current mRNA
expression screens such as the BD
Atlas Array or the BD
Riboscreen Membrane Array
Avoids the limitations of traditional protein screening techniques
such as 2-D Gel Electrophoresis
and Mass Spectrometry
Utilizes highly specific and sensitive monoclonal antibodies detecting sub-nanogram levels of protein
Carefully formulated antibody
combinations yield reproducible,
semi-quantifiable results
Immediate identification of proteins with altered expression
Reagents for follow-up studies are
well characterized and readily
available
BD PowerBlot in Action
Protein expression changes in SY5Y cells after exposure to NGF or staurosporine
Control
Lane 5
NGF
10
15
20
25
30
35
40
Control
Lane 5
Lane 5
10
15
20
25
30
35
40
15
20
25
30
35
40
Staurosporine
10
15
20
25
30
35
40
Lane 5
10
a AF6
182 kDa
b BM28
125 kDa
c PKC
78 kDa
d Janusin
180 kDa
e GDNFR
58 kDa
f Caspase-7
35 kDa
g PP1
36 kDa
h CarboxyPeptidase E
50 kDa
i Raft1
245/200 kDa
j N-Copine
62 kDa
Western blot images from Template A of the BD PowerBlot analysis. This template screens 183 of the 820 different antibody specificities. a thru j show various protein signals that significantly changed after exposure to NGF (100 ng/ml for 24 h) and/or staurosporine (500 nM for 16 h).
32
www.bdbiosciences.com
The Method
1. Lysed sample
2. SDS-PAGE
3. Transfer
6. Data analysis
5. Data capture
4. Antibody incubation
Membranes containing
samples are probed with
over 900 mAbs.
7. Data summary
8. Bioinformatics
A summary file (not shown) is provided which
lists all protein expression changes detected, in
order of confidence, 1 through 5, with 5 being
the highest confidence. The confidence level is
based on fold change, reproducibility, and signal intensity.
Level 5 - changes greater than 2-fold in triplicate from good quality signals.
Level 4 - changes 1.50-1.99-fold in triplicate
from good quality signals.
Level 3 - changes 1.25-1.49-fold in triplicate
from good quality signals.
Level 2 - changes greater than 1.25-fold in triplicate from low signals.
Data is exported to an electronic spreadsheet that indicates raw signal intensity data, normalized data,
and comparative analysis.
Level 1 - changes greater than 2-fold in duplicate from good quality signals.
Upon completion of the BD PowerBlot service, a results package is assembled for you, which includes the catalog numbers for all the antibodies used. If youre unsure
of the biological relevance of any of the proteins evaluated, a quick visit to our website (www.bdpowerblot.com) and a search by catalog number will yield a pdf file of
the technical document associated with your protein of interest.
www.bdbiosciences.com
33
1 hr
4 hr
8 hr
Custom BD PowerBlot
Data
In this experiment, a customer used
the Custom BD PowerBlot screening
service to identify nuclear and/or
mitochondrial
markers
that
remained unchanged during staurosporine-induced apoptosis in
Jurkat cells. Control, 1-, 4-, and 8-hr
staurosporine-treated
lysates
(Jurkat+Staurosporine
apoptosis
lysate kit, Cat. No. 611626) were
loaded on gels and transferred to
PVDF membranes. A variety of anti-
Results
This Custom BD PowerBlot analysi
in fig. 1 shows decreases in the
expression of several proteins that
are degraded during apoptosis
(PARP, DFF45, Caspase-8, and
Caspase-7). Conversely, expression
of several proteins remained
unchanged, such as the RNA Pol. III
transcription termination factor La
Protein, the regulator of chromosome condensation RCC1, the mitochondrial enzyme Methyl-malonylCoA mutase, and the heat shock protein Hsp70. These may all be good
candidates for use as protein-loading
normalization controls in studies of
apoptosis. Interestingly, one of the
nuclear factors tested, TBP, was dramatically upregulated after staurosporine exposure. Thus, TBP may
play a key role in the transcriptional
regulation of genes involved in apoptosis.
BD PowerBlot Citations
1. Cunningham, B.A. 2001. Now Thats Service. The Scientist. 14(19). www.the-scientist.com.
2. Castedo M, Ferri KF, Blanco J, Roumier T, Larochette N, Barretina J, Amendola A, Nardacci R, Metivier D, Este JA, Piacentini
M, Kroemer G. 2001. Human immunodeficiency virus 1 envelope glycoprotein complex-induced apoptosis involves mammalian target of rapamycin/FKBP12-rapamycin-associated protein-mediated p53 phosphorylation. J Exp Med. 194(8):1097110.
3. Arnoult D, Tatischeff I, Estaquier J, Girard M, Sureau F, Tissier JP, Grodet A, Dellinger M, Traincard F, Kahn A, Ameisen JC,
Petit PX. 2001. On the evolutionary conservation of the cell death pathway: mitochondrial release of an apoptosis-inducing
factor during Dictyostelium discoideum cell death. Mol Biol Cell. 12(10):3016-30.
34
www.bdbiosciences.com
Untreated
99%
M1
95%
Relative Cell Number
Untreated
240
160
80
M1
120
5%
M2
+ Camptothecin
untreated
untreated
kD
Mouse Thymocytes
kD
36
31
36
31
ProCaspase-3
1%
M2
Liver (mouse)
21
Active
14
21
Active
Caspase-3
14
6
6
C
Treated with
Camptothecin
(4 hr)
160
48%
80
M1
D
240
55%
M1
120
52%
M2
Treated with
mouse Fas mAb,
clone Jo2 (6 hr)
45%
M2
100
101
102
103
100
101
Immunoprecipitation;
pAb, Cat. No. 67341A
Western Blot;
pAb, Cat. No. 65906E
Western Blot;
pAb, Cat. No. 65906E
102
103
Log Fluorescence
Flow cytometric analysis of apoptotic and non-apoptotic populations using antiactive caspase-3 antibodies. Jurkat T cells (A, C) or mouse thymocytes (B, D) were left
untreated (A, B) or treated for 4 hr with camptothecin (C) or 6 hr with a mouse Fas
monoclonal antibody, clone Jo2 (Cat. No. 554254) (D) to induce apoptosis. Cells were
stained with PE-conjugated active caspase-3 antibodies (Cat. No. 557091). Untreated
cells were primarily negative for the presence of active-caspase-3, whereas about
half of the population of cells induced to undergo apoptosis had detectable caspase3 activity.
References:
1. Nicholson, D.W., A. Ali, N.A. Thornberry, J.P. Vaillancourt, C.K. Ding, M. Gallant, Y. Gareau, P.R. Griffin, M. Labelle and M. Lazebnik. 1995. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376:37-43.
2. Patel, T., G.J. Gores and S.H. Kaufmann. 1996. The role of proteases during apop-tosis. FASEB J. 10:587-597.
www.bdbiosciences.com
35
Lysate Treatment
Camptothecin (4M)
1.2
Staurosporine (4M)
Control
1.0
Blank
OD 450nm
0.8
0.6
0.4
0.2
0
0.125
0.063
0.031
0.016
0.008
E
anti species
enzyme-labeled
antibody
detection
antibody
3.5
antigen
OD 450nm
3.0
capture
antibody
2.5
2.0
1.0
0.5
0.0
0
Staur (4M)
Control
Blank
Staur (4M)
Control
Blank
0.8
OD 450nm
1.5
Jurkat
Mcf-7
0.6
0.4
0.2
0
0.125
0.016
0.063
0.031
0.008
36
www.bdbiosciences.com
Product Listing
Description
Clone(s)
Size
Cat. No.
Fas
Caspase-3/CPP32
Caspase-3/CPP32
Caspase-3
Caspase-3, Active
Jo2
19
19
Polyclonal
Polyclonal
Ms
Caspase-3, Active
C92-605
C92-605
BD Pharmingen
BD Transduction Laboratories
Hu, Ms
IP,FC,FA
WB, IF
WB, IF
WB, IHC(P)
IP,IHC(F),FC
IHCF
FC
FC, IF, IP
FC
FC
FC
FC
NA/LE
Purified
Purified
Serum
Purified
Biotin
PE
Purified
Biotin
FITC
PE
Kit
0.5 mg
50 g
150 g
0.1 ml
0.1 mg
0.1 mg
100 tests
25 g
100 tests
100 tests
100 tests
100 tests
15400D
611320
611321
65906E
67341A
67342A
67345X
68651G
68652X
68654X
68655X
6976KK
554254
611320
611321
556425
557035
557038
557091
559565
550557
559341
550821
550480
C92-605
Hu, Ms
FC
Kit
100 tests
6865KK
550822
Polyclonal
Hu, Ms
FC
Kit
100 tests
6899KK
559762
19 & C92-605
Hu, Ms
ELISA
ELISA Pair
69941K
550578
19 & C92-605
19
19
46
46
Hu,
Hu,
Hu,
Hu,
Hu,
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA Set
HRPO
HRPO
Rec. Prot.
Rec. Prot.
8072KK
C31725-050
C31725-150
C76920-050
C76920-150
550930
610324
610325
611048
611049
Hu, Ms
Ms
Ms
Ms
Ms
Ms
50 g
150 g
50 g
150 g
www.bdbiosciences.com
37
caspase-3 are added to the bead mixture and the resulting reactions are
measured by fluorescent intensity
using a flow cytometer. The kit also
contains lyophilized apoptotic cell
lysate for generating active caspase-3
standard curves, which enables relative quantification of active caspase3 in the researchers cell lysate
samples. Heretofore, caspase-3 in
cell lysates has been typically analyzed by classical immunoprecipitation and western blot technologies.
These technologies are the most popular for analyzing intracellular proteins in cell lysates. Quantification is
generally restricted to such descriptors as detected or not detected, up
or down regulated, or fold changes
(e.g. two to ten fold). The Human
Active Caspase-3 CBA Kit offers an
104
Wash
Control
Camptothecin
MFI
103
102
Analyze
10
Pro caspase-3
Active caspase-3
Other proteins
Capture mAb/Bead:
Pro/Active caspase-3
Detection mAb:
Active caspase-3PE
10
102
Size
Cat. No.
100 tests
1 CD
552124
550065
www.bdbiosciences.com
104
P the
t iActive-Caspase-3
( )
Representative standard curve data from
CBA Kit. Standard
curves were generated from apoptotic and control Jurkat cell lysate. Jurkat cell cultures
were treated with 1 M camptothecin for 4 hr to induce apoptosis. Control cell cultures
were left untreated. Cell lysates were lyophilized, reconstituted, and serially diluted. The
data shows that active caspase-3 could be measured over a two log dynamic range in
apoptotic lysates. Control cells had minimal caspase-3 activity compared with apoptotic
cells.
Description
38
103
BD Pharmingen
References
1. Cohen, G. M. 1997 Caspases: the executioners of apoptosis. Biochem J. 326:116.
2. Lazebnik Y A, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC. 1994: Cleavage of poly(ADP-ribose) Polymerase by
a proteinase with properties like ICE. Nature 371(6495):346-7.
3. Porter, A. G. & Janicke, R. U. 1999 Emerging roles of caspase-3 in apoptosis. Cell Death Diff. 6:99104.
4. Zou, H., Li, Y., Liu, X. & Wang, X. 1999 An APAF-1 cytochrome c multimeric complex is a functional apoptosome that
activates procaspase-9. J. Biol. Chem. 274:1154911556.
5. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahnad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-depedent
formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91(4):479-89.
www.bdbiosciences.com
39
DEVD-pNA
pNA
+ DEVD
Caspase-3
DEVD-AFC
IETD-pNA
AFC
pNA
+ DEVD
+ IETD
Caspase-8
IETD-AFC
AFC
+ IETD
Jurkat cells
Fluorescence units
2,500
2,000
1,500
1,000
500
+ Fas Ab
+ caspase-6 inh.
+ Fas Ab
+ caspase-9 inh.
+ Fas Ab
Fas Ab
NIH/3T3 cells
600
Caspase-3
+ Ac-DEVD-AFC
Caspase-6
+ Ac-VEID-AFC
Caspase-7
+ Ac-DEVD-AFC
A1
Caspase-3
+ Ac-DEVD-AFC
+ Ac-DEVD-CHO
B1
Caspase-6
+ Ac-VEID-AFC
+ Ac-VEID-CHO
C1
Caspase-7
+ Ac-DEVD-AFC
+ Ac-DEVD-CHO
800
600
400
200
+ Staurosporine
+ caspase-6 inh.
+ Staurosporine
+ caspase-9 inh.
+ Staurosporine
Staurosporine
1,000
Fluorescence units
400
200
0
600
400
200
0
450
550 450
550 450
(nm)
550
40
www.bdbiosciences.com
Product Listing
Description
Format/Conc.
Apps.
Size
Cat. No.
556485
Caspase-3
Kit
SF
100 tests
5x200g
5x200g
50ml
50ml
50ml
6632KK
Kit
FA
25 assays
100 assays
K2026-1
K2026-2
Kit
FA
25 assays
100 assays
K2027-1
K2027-2
Set
FA,SF
20 tests
5 g
200 g
20 g
6628KK
556473
556476
Caspase-6
Set
FA,SF
20 tests
5 g
200 g
20 g
6629KK
Kit
FA
25 assays
100 assays
K2015-1
K2015-2
Set
FA,SF
20 tests
5 g
200 g
20 g
6630KK
Kit
FA
25 assays
100 assays
K2028-1
K2028-2
Kit
FA
25 assays
200 assays
K2029-1
K2029-2
Set
FA,SF
20 tests
5 g
200 g
20 g
6631KK
Caspase-7
556479
Caspase-8
BD Pharmingen
BD Clontech
556482
5 g
200 g
20 g
www.bdbiosciences.com
41
Product Listing
Description
Format/Conc.
Apps.
Size
Cat. No.
Kit
FA
25 assays
100 assays
K2015-1
K2015-2
Enzyme
FA
Enzyme
FA
Enzyme
FA
Enzyme
FA
5 g
10 g
5 g
10 g
5 g
10 g
5 g
10 g
66281V
66281T
66291V
66291T
66301V
66301T
66311V
66311T
Caspase-8
556472
556471
556475
556474
556478
556477
556481
556480
1 mM
SF
1 mM
SF
SF
SF
1 mM
1 mM
SF
SF
SF
SF
1 mM
100 l
1 mg
100 l
1 mg
1 mg
1 mg
100 l
100 l
1 mg
1 mg
1 mg
1 mg
100 l
66091U
67201U
66081U
66221U
66941U
66951U
66961U
66971U
8173-1
556451
8171-1
556574
556449
556465
8170-1
8172-1
556548
556550
556552
556554
8174-1
Apoptosis Inducer
Human TNF-
10 g
8157-1
AFC (7-amino-4-trifluoromethyl coumarin) emits a yellow-green fluorescence at 505 nm if excited at 400 nm after the substrate is cleaved by the protease.
AMC (7-amino-4-methoxy coumarin) fluorescence can be measured using a 380-nm excitation filter and a 460-nm emission, upon substrate cleavage.
Fluorescence detection is highly sensitive and can be used to measure even very small amounts of active caspase.
The caspase colorimetric assay measures the proteolytic cleavage of the chromophore p-nitroanilide (pNA) by caspases.
Liberated pNA can be monitored colorimetrically by absorbance at 405 nm.
When coupled to an aldehyde group (CHO) or fluoromethylketone (fmk), tetrapeptides function as potent inhibitors of caspase activity and can be used
to block caspase-mediated cleavage of the corresponding fluorogenic substrates.
ApoAlert Caspase Fluorescent Assay Kits were cited in the following articles:
Jingyu Diao, Aye Aye Khine , Farida Sarangi, Eric Hsu, Caterina Iorio, Lee Anne Tibbles, James R. Woodgett, Josef Penninger, and Christopher D. Richardson. 2001. X Protein of Hepatitis B
Virus Inhibits Fas-mediated Apoptosis and Is Associated with Up-regulation of the SAPK/JNK Pathway. J. Biol. Chem. 276(11):8328-8340
Saadi Ghatan, Stephen Larner, Yoshito Kinoshita, Michal Hetman, Leena Patel, Zhengui Xia, Richard J. Youle, and Richard S. Morrison. 2000. p38 MAP Kinase Mediates Bax Translocation
in Nitric Oxideinduced Apoptosis in Neurons. J. Cell Biol. 150(2):335347
Narvaez CJ, Welsh J.2001. Role of mitochondria and caspases in vitamin D-mediated apoptosis of MCF-7 breast cancer cells. J Biol Chem.;276(12):9101-7.
Delphine Lechardeur, Luke Drzymala, Manu Sharma, Danuta Zylka, Robert Kinach, Joanna Pacia, Christopher Hicks, Nawaid Usmani, Johanna M. Rommens, and Gergely L. Lukacs. 2000.
Determinants of the Nuclear Localization of the Heterodimeric DNA Fragmentation Factor (ICAD/CAD) J. Cell Biol. 150(2):321334
ApoAlert Caspase Colorimetric Assay Kits were cited in the following articles:
Shasi V. Kalivendi, Srigiridhar Kotamraju, Hongtao Zhao, Joy Joseph, and B. Kalyanaraman. 2001. Doxorubicin-induced Apoptosis Is Associated with Increased Transcription of Endothelial
Nitric-oxide Synthase. J. Biol. Chem. 276(50):47266-47276
Yoshio Tomizawa, Yoshitaka Sekido, Masashi Kondo, Boning Gao, Jun Yokota, Joelle Roche, Harry Drabkin, Michael I. Lerman, Adi F. Gazdar, and John D. Minna. 2001. Inhibition of lung
cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B. Proc. Natl. Acad. Sci. +
42
www.bdbiosciences.com
BD Pharmingen
BD Clontech
EYFP NLS
NES DEVD
Nucleus
Figure 1.
Description
Size
Cat. No.
pCaspase3-Sensor Vector
20 g
8185-1
BD Clontech
www.bdbiosciences.com
43
EYFP NLS
A ready-to-use fluorescent
reporter
By transfecting cells with pDsRed2Bid or pd4EGFP-Bid, you can easily
detect this translocation using conventional fluorescence microscopy
(Figure 1)*. And because you can
examine individual cells, you can
design cotransfection and multicolor
experiments to study the effects
other proteins have on this crucial
apoptotic event. Once you identify a
proteins pro- or anti-apoptotic
property, you can go on to map the
domains responsible for inducing or
blocking the Bid-dependent pathway.
Additionally, by visualizing Bid in
conjunction with appropriate cellcycle indicators, you can investigate
the potential cell cycle dependence of
Bid translocation. Assays that detect
apoptosis in bulk populations cannot
provide this information. Now you
can dissect the Bid-dependent pathway in an easy non-invasive manner.
44
www.bdbiosciences.com
Untreated
Treated
Figure 1. Monitoring Bid activation with the pDsRed2- Bid and pd4EGFP-Bid
Vectors. 3T3 cells were transiently transfected with either pDsRed2-Bid (Panels A & B) or
pd4EGFP-Bid (Panels C & D). Approximately 24 hr later, cells were incubated with 700 nM
staurosporine, an apoptosis inducing agent, at 37C for 3 hr. Cells were then fixed and
examined with a Zeiss Axioskop equipped with the appropriate light filters. In untreated
cells (Panels A & C), the Bid reporters (Bid-DsRed2 and Bid-d4EGFP) distribute uniformly in
the cytosol, as expected. Following induction (Panels B & D), the reporters translocate to
the surface of mitochondria, forming intensely fluorescent clustersclear evidence that
the Bid pathway has been activated.
Description
Size
Cat. No.
pDsRed2-Bid Vector
pd4EGFP-Bid Vector
20 g
20 g
6977-1
6979-1
References
1. Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH. 2000. Pro-apoptotic cascade activates BID, which
oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ. 7(12):1166-73. Review.
2. Living Colors DsRed2 (July 2001) CLONTECHniques XVI(3):2-3
3. Li X, Zhao X, Fang Y, Jiang X, Duong T, Fan C, Huang CC, Kain SR. 1998. Generation of destabilized green fluorescent
protein as a transcription reporter. J Biol Chem 273(52):34970-5.
4. Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S, Maundrell K, Antonsson B, Martinou JC.1999.
Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell
Biol.144(5):891-901.
5. Zha J, Weiler S, Oh KJ, Wei MC, Korsmeyer SJ. 2000. Posttranslational N-myristoylation of BID as a molecular switch for
targeting mitochondria and apoptosis. Science. 290(5497):1761-5.
6. Lutter M, Fang M, Luo X, Nishijima M, Xie X, Wang X. 2000. Cardiolipin provides specificity for targeting of tBid to mitochondria. Nat Cell Biol. 2(10):754-61.
BD Clontech
www.bdbiosciences.com
45
Fluorescence units
3,500
3,000
2,500
Control
1 min UV
5 min UV
15 min UV
2,000
1,500
Induce
apoptosis
1,000
500
0
0
1 M Staurosporine
3,500
Fluorescence units
BD
Biosciences
Clontech
ApoAlert Glutathione Detection
Kit is a quantitative in vitro assay
that detects decreased cytosolic glutathione (GSH) levels that occur
early in apoptosis in some cell types.
In healthy cells, glutathione acts as a
redox buffer. However, in Jurkat and
some other cell types, the plasma
membrane contains an ATP-dependent GSH transport system that is
triggered by the initiation of apoptosis. When GSH is actively pumped
out of the cell, the cytosol is shifted
from a reducing to an oxidizing
environment1.
The
ApoAlert
Glutathione Detection Kit includes a
monochlorobimane (MCB) dye that
fluoresces blue when bound to glutathione.
The
decrease
in
gluthathione levels between nonapoptotic and apoptotic cell extracts
is easily detected using a fluorometer
or 96-well fluorometric plate reader.
UV Irradiation
3,000
2,500
2,000
1,500
1,000
Control
+ Staurosporine [1 M]
500
0
0
MCB
Fluorescence units
3,500
3,000
2,500
2,000
1,500
MCB
1,000
GSH
Control
+ Fas mAb [500 ng/ml]
500
0
0
Description
Format
Apps.
Size
Cat. No.
Kit
FA, SF
25 assays
100 assays
K2014-1
K2014-2
References
1.van den Dobbelsteen DJ, Nobel CS, Schlegel J, Cotgreave IA, Orrenius S, Slater AF. 1996. Rapid and specific efflux of reduced glutathione during apoptosis induced by anti-Fas/APO-1 antibody. J Biol Chem. 271(26):15420-7.
46
www.bdbiosciences.com
BD Clontech
A
Fraction:
Mito
kDa
Cyt
Cyt
Cyt
Mito
kDa
kDa
43
43
43
Mito
29
29
29
18
18
14
cytochrome c
COX4
18
14
14
Induction:
Description
Format
Apps.
Size
Cat. No.
Kit
FA, SF
25 assays
100 assays
K2014-1
K2014-2
References
1. Liu X, Kim CN, Yang J, Jemmerson R, Wang X. 1996 Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell. 12;86(1):147-57.
2. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, Wang X. 1997. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic
protease cascade. Cell. 91(4):479-89.
BD Clontech
www.bdbiosciences.com
47
700
Apoptotic
Nonapoptotic
Cells
560
420
280
140
0
100
101
102
103
104
Green fluorescence
Figure 1. MitoSensor staining of HeLa cells. Panel A. HeLa cells were rinsed with serum-free media, stained with
MitoSensor at 37C for 20 min, rinsed once with incubation buffer, and analyzed by fluorescence microscopy using a bandpass filter. Non-apoptotic cells exhibit an intense red fluorescence. Panel B. Cells were treated with 1 M staurosporine prior
to staining with MitoSensor. Apoptotic cells exhibit a bright green fluorescence.
Description
Format
Apps.
Size
Cat. No.
Kit
FA, FC
100 assays
K2017-1
References
1.Green DR, Reed JC.1998. Mitochondria and apoptosis. Science.281(5381):1309-12. Review.
The ApoAlert Mitochondrial Membrane Sensor Kit was cited in the following articles:
Biswal SS, Datta K, Shaw SD, Feng X, Robertson JD, Kehrer JP. 2000. Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis
in lipoxygenase-deficient FL5.12 cells. Toxicol Sci.53(1):77-83.
Donnelly ET, O'Connell M, McClure N, Lewis SE. 2000. Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa. Hum
Reprod.15(7):1552-61.
Zhu J, Chen X. 2000 MCG10, a novel p53 target gene that encodes a KH domain RNA-binding protein, is capable of inducing apoptosis and cell cycle arrest in G(2)-M. Mol Cell Biol.
20(15):5602-18.
48
www.bdbiosciences.com
BD Clontech
101
102
103
103 104
102
101
Propidium Iodide
104
100
Annexin V-FITC
M2
100
101
102
101
102
103
104
Annexin V-FITC
M1
Treated with
Human Fas
mAb
Cell Frequency
100
M1
100
103 104
101
102
B Treated with
Human Fas
mAb
100
Cell Frequency
Propidium Iodide
A
A Untreated
Treated with
Human Fas
mAb
+ Purified Annexin V
M1
M1
M2
M2
M2
103
104
100
101
102
103
104
Annexin V-FITC
Flow cytometric analysis of apoptotic cells using Annexin V-FITC. HBP-ALL leukemia
cells were left untreated (A) or treated for 2 hr (B) with anti-human Fas antibody, clone DX2
(Cat. No. 555670) and Protein G*. Cells were incubated with Annexin V-FITC (Cat. No.
65874X or 556419) in a buffer containing Propidium iodide (PI), (Cat. No. 556463) and analyzed by flow cytometry. Untreated cells were primarily Annexin V-FITC and PI negative (inset
A), indicating that they were viable and not undergoing apoptosis. After a 2 hr treatment
with DX2, the majority of cells were undergoing apoptosis (Annexin V-FITC positive and PI
negative; inset B). The M1 and M2 gates demarcate Annexin V-FITC negative and positive
populations, respectively.
100
101
102
103
104
100
101
102
103
104
Annexin V-FITC
Flow cytometric analysis of Annexin V-FITC staining and blocking with recombinant
Annexin V. Jurkat T cells were induced to undergo apoptosis by treatment with anti-human
Fas antibody, clone DX2 (Cat. No. 555670) and Protein G* for 3 hr. Cells were then incubated with Annexin V-FITC (Cat. No. 556420 or 556419) alone (A) or with Annexin V-FITC
in the presence of recombinant Annexin V (Cat. No. 556416) (B) to block Annexin V-FITC
binding sites, thus demonstrating the specificity of Annexin V-FITC staining. *The addition of
Protein G enhances the ability of DX2 to induce apoptosis, presumably by cross-linking Fas
receptors.
www.bdbiosciences.com
49
Cell Frequency
Annexin V-PE
M2
M1
M2
M1
M2
M1
M2
M1
M2
M1
0h
M2
M1
M2
M1
M2
M1
1h
2h
Control
M2
M1
M2
M1
3h
4h
Camptothecin-treated
Comparison of Annexin-V and Active Caspase-3 Assays: The Annexin V assay is perhaps the most widely used assay today,
facilitated by the observation that PS translocation appears to be a universal apoptotic phenomenon. It has been detected in
mammalian, insect, and plant cells under the action of most, if not all, triggers of apoptosis. Although the Annexin V and
Active Caspase-3 assays are based on different biological phenomena, they are both used to identify cells that are in the early
stages of apoptosis .
200
Counts
160
120
uninduced
induced with
anti-Fas
80
40
0
100
101
102
103
EGFP binding
Figure 1. ApoAlertTM Annexin V-EGFP generates a bright green fluorescent signal. Jurkat cells were incubated with 200
ng/ml human fas monoclonal antibody (clone CH-11) for 8 hr to induce apoptosis. Cells were cen-trifuged, resuspended in
1X Binding Buffer, and incubated with 1 ml of Annexin V-EGFP for 5 min in the dark. Panel A. Detection of Annexin V-EGFP
by fluorescence microscopy. Positive cells exhibit green fluorescence around the plasma membrane. Panel B. Detection of
Annexin V-EGFP by flow cytometry.
Notice to Purchaser
The Annexin V-EGFP reagent is covered by U.S. Patents #5,491,084
and 5,066,787, and is the subject of pending patent applications. All
purchasers are granted an automatic license with the purchase of the
Annexin V-EGFP reagent to use it for internal research purposes.
However, this license excludes specifically any right to modify the
reagent for resale, to use the reagent for the manufacture of commercial products, or to use the reagent in humans or for diagnostic
purposes. Certain Living Colors products, and other products containing "enhanced" GFP variants, are covered by the following license
terms. Use of CLONTECH's Living Colors products containing DNA
sequences coding for mutant Aequorea victoria green fluorescent protein (GFP) variants or proteins thereof requires a license from Aurora
Biosciences Corporation under U.S. Patent Nos. 5,625,048, 5,777,079,
6,054,32 and 5,804,387 and other pending U.S. and foreign patent
applications. In addition, certain CLONTECH products are made under
U.S. Patent No. 5,804,387 licensed from Stanford University. Not-For-
50
www.bdbiosciences.com
Active
Caspase-3-FITC
Product Listing
Description
Format/Conc.
Applications Size
Cat. No.
Unlabeled
Biotin
Biotin
FITC
FITC
FITC
PE
PE
APC
APC
Cy5
Cy5
Cy5.5
Cy5.5
EGFP tagged
Kit
Kit
Kit
Kit
Kit
Kit
Buffer
Buffer
Buffer
Buffer
65871A
65872H
65872X
65874H
65874X
Buffer
Kit
FC
FC
FC
FC
FC
FC, FM
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC
FC, FM
FC, FM
FC
FC
FC
FC, FM
FC
FC
FC
FC
FM
556416
556417
556418
556419
556420
8133-1
556421
556422
550475
550474
559934
559933
559936
559935
8137-1
556547
556570
K2025-1
K2025-2
K2019-1
K2019-2
556454
8134-1
556463
555816
555815
559925
550911
0.1 mg
200 tests
100 tests
200 tests
100 tests
500 assays
200 tests
100 tests
200 tests
100 tests
200 tests
100 tests
200 tests
100 tests
500 assays
100 tests
100 tests
50 assays
200 assays
50 assays
200 assays
50 ml
100 ml
2.0 ml
100 tests
500 tests
2ml
65875H
65875X
65879H
65879X
6587MH
6587MX
6587NH
6587NX
6693KK
6710KK
66121E
66211E
34321X
34321J
68981E
8065KK
Annexin V-FITC Detection Kit I, ApoAlert Annexin V-FITC Apoptosis Kit and ApoAlert Annexin V-EGFP Apoptosis Kit
Kit Components
Annexin V-FITC or Annexin V-EGFP
Binding Buffer
Propidium Iodide
User Manual
The ApoAlert Annexin V-FITC Apoptosis Kit was cited in the following articles:
Takao N, Mori R, Kato H, Shinohara A, Yamamoto K. 2000. c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated functions in chromosomal maintenance. J Biol Chem.
275(2):725-8.
Maeshima Y, Colorado PC, Torre A, Holthaus KA, Grunkemeyer JA, Ericksen MB, Hopfer H, Xiao Y, Stillman IE, Kalluri R. 2000. Distinct antitumor properties of a type IV collagen domain
derived from basement membrane. J Biol Chem. 275(28):21340-8.
Persad S, Attwell S, Gray V, Delcommenne M, Troussard A, Sanghera J, Dedhar S. 2000. Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell
cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci U S A.97(7):3207-12.
The ApoAlert Annexin V-EGFP Apoptosis Kit was cited in the following articles:
Shaoqiong Chen, Denis C. Guttridge, Zongbing You, Zhaochen Zhang, Andrew Fribley, Marty W. Mayo, Jan Kitajewski, and Cun-Yu Wang. 2001. Wnt-1 Signaling Inhibits Apoptosis by
Activating b-Catenin/T Cell Factormediated Transcription. J. Cell Biol. 152(1):8796
Yi Ting Zhou, Unice J.K. Soh, Xun Shang, Graeme R. Guy and Boon Chuan Low. 2002. The BNIP-2 and Cdc42GAP Homology / Sec-14p-like domain of BNIP-Sa a is a novel apoptosis-inducing sequence. J. Biol. Chem. In press
BD Pharmingen
BD Clontech
www.bdbiosciences.com
51
A simple fluorimetric
method for measuring
nitric oxide in apoptotic
cells
Simultaneous detection of
apoptotic events
52
www.bdbiosciences.com
Simple fluorescence-based
assay
It is easy to prepare cells for the
NO/Annexin V assay (Figure 1). The
assay is nonenzymatic, requires no
fixatives, and is suitable for adherent or non-adherent cells. First, incubate cells with the NO-sensing dye
for 30 minutesno washing or rinsing steps are requiredthen induce
apoptosis using your method of
choice. You can assay nitric oxide
and PS externalization separately or
together. In either case, simply collect
cells at preselected intervals, resuspend in buffer (with or without
annexin V-phycoerythrin) and analyze by flow cytometry.
Annexin V
PE
Resuspend in
Binding Buffer
Incubate for
15 min at RT
FACS
FACS
(FL-1)
Figure 1. Measuring nitric oxide in cultured cells. The NO Sensor Dye (Excitation max 495 nm; Emission max 515 nm) fluoresces approximately 100 times brighter after binding nitric oxide5. Although the dye readily enters cells, it can not exitcellular esterases lock the dye inside by removing its hydrophobic tail. Annexin V-phycoerythrin (PE) binds phosphatidylserine
when it flips to the outer leaflet of the plasma membrane, a hallmark of apoptotic cells.
www.bdbiosciences.com
53
6 hr
UV
+ UV
UV
+ UV
6 hr
24 hr
B
B
6 hr
24 hr
24 hr
Figure 3. UV light causes a surge in nitric oxide synthesis. Cell cultures were incubated with the NO Sensor Dye (5 M)
for 30 min, exposed to UV light for 5 min, and then incubated at 37C for the indicated times. Green fluorescence was
measured by flow cytometry. Panel A. HeLa cells. Panel B. Jurkat cells. 3T3 cells were also tested with similar results (not
shown).
Description
Assays
Cat. No.
25
100
K2013-1
K2013-2
Components
Nitric Oxide Sensor Dye
Annexin V-Phycoerythrin (PE)
Annexin V Binding Buffer
References
1. Ushmorov A, Ratter F, Lehmann V, Droge W, Schirrmacher V, Umansky V. 1999. Nitric-oxide-induced apoptosis in human
leukemic lines requires mitochondrial lipid degradation and cytochrome C release. Blood. 93(7):2342-52
2. Murphy MP. 1999. Nitric oxide and cell death. Biochim Biophys Acta. 1411(2-3):401-14. Review.
3. Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, Maeda D, Imai Y, Irimura T, Nagano T. 1998. Direct evidence of
nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS
Lett. 427(2):263-6.
4. Kojima H, Sakurai K, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. 1998. Development of a fluorescent indicator for nitric oxide based on the fluorescein chromophore. Chem Pharm Bull (Tokyo).46(2):373-5.
5. Kojima H, Nakatsubo N, Kikuchi K, Kawahara S, Kirino Y, Nagoshi H, Hirata Y, Nagano T. 1998. Detection and imaging of
nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal Chem. 70(13):2446-53.
54
www.bdbiosciences.com
BD Clontech
APO-BRDU Assay
M1
M2
100
101
10 2
B
Relative Cell Number
APO-BRDU*
M2
10
10
10
55
10
10
Treated with
Human Fas
mAb (12 hr)
M1 = 34%
M2 = 66%
M1
M2
M1
104
M1
10
References
1. Enari, M., H. Sakahira, H. Yokoyama, K. Okawa, A. Iwamatsu and S. Nagata. 1998. A caspase-activated DNase that
degrades DNA during apoptosis, and its inhibitor ICAD. Nature. 394:43-50.
2. Sakahira, H., M. Enari and S. Nagata. 1998. Cleavage of CAD inhibitor in CAD activation and DNA degradation during
apoptosis. Nature. 391:96-99
3. Walker, R.P., L. Kokileva, J. LeBlanc and M. Sikorska. 1993. Detection of the initial stages of DNA fragmentation in apoptosis. Biotechniques 15:1032-1036.
4. Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami and T. Traganos, T. 1997. Cytometry in cell necrobiology:
Analysis of apoptosis and accidental cell death (necrosis). Cytometry 27:1-20
5. Li, X., F. Traganos, M.R. Melamed, and Z. Darzynkiewicz. 1995. Single step procedure for labeling DNA strand breaks
with fluorescein- or BODIPY-conjugated deoxynucleotides. Detection of apoptosis and bromodeoxyuridine incorporation.
Cytometry 20:172-180.
6. Li, X. and A. Darzynkiewicz. 1995. Labelling DNA strand breaks with Brd-UTP. Detection of apoptosis and cell proliferation Cell Prolif. 28:572-579
10 3
Treated with
Human Fas
mAb (2 hr)
M1 = 63%
M2 = 37%
APO-DIRECT*
www.bdbiosciences.com
Untreated
M1 = 99%
M2 = 1%
10
10
BrdU-FITC
10
10
50
Counts
50
Counts
40
30
20
40
30
20
10
10
100
101
103
102
FITC Fluorescence Intensity
104
100
101
102
103
FITC Fluorescence Intensity
104
Figure 2. FACS analysis of DNA fragmentation in apoptotic cells in suspension. Jurkat cells were grown in RPMI 1640
medium + 10% FBS. Apoptosis was induced by treatment with 200 ng/ml anti-Fas mAb for 15.5 hr. The ApoAlert DNA fragmentation assay was performed according to the User Manual. Cells were analyzed by flow cytometry. Panel A. Uninduced
cells. Panel B. Induced cells.
56
www.bdbiosciences.com
Product Listing
Description
Apps.
Format
Size
Cat. No.
APO-BRDU KIT
Fluorescein Labeled Anti-BrdU mAb
PI/Rnase Staining Buffer
Reaction, Rinsing and Wash Buffer
Br-dUTP
Negative/Positive Control Cells
TdT Enzyme
FC
Kit
60 tests
6576KK
556405
APO-DIRECT KIT
PI/Rnase Staining Buffer
Reaction, Rinsing and Wash Buffer
FITC-dUTP
Negative/Positive Control Cells
TdT Enzyme
FC
Kit
50 tests
6536KK
556381
IF, FC
Kit
25 assays
100 assays
K2024-1
K2024-2
Equilibration Buffer
Nucleotide Mix
TdT Enzyme
SSC
Proteinase K
Plastic Coverslips
References
1. Gavrieli Y, Sherman Y, Ben-Sasson SA. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol. Nov;119(3):493-501.
The ApoAlert DNA Fragmentation Assay Kit was cited in the following articles:
Christelle Forcet, Xin Ye, Laure Granger, Vronique Corset, Hwain Shin, Dale E. Bredesen, and Patrick Mehlen. 2001. The dependence receptor DCC (deleted in colorectal cancer) defines an
alternative mechanism for caspase activation. Proc. Natl. Acad. Sci. 98(6):3416-3421
Prasad Devarajan, Maryely De Leon, Farahnaz Talasazan, Alan R. Schoenfeld , Eliot J. Davidowitz , and Robert D. Burk. 2001. The von Hippel-Lindau Gene Product Inhibits Renal Cell
Apoptosis via Bcl-2-dependent Pathways. J. Biol. Chem. 276( 44):4059940605
Srigiridhar Kotamraju, Neil Hogg, Joy Joseph, Larry K. Keefer, and B. Kalyanaraman. 2001. Inhibition of Oxidized Low-density Lipoprotein-induced Apoptosis in Endothelial Cells by Nitric
Oxide J. Biol. Chem. 276(20):17316-17323
BD Pharmingen
BD Clontech
www.bdbiosciences.com
57
24-mer
HOHO-
5'
3'
-OH HO-P
HO5'
12-mer
3'
-OH
Fill in ends
800
600
400
200
Figure 1. The ApoAlert LM-PCR assay. Apoptosis was induced in Jurkat cells by incubation with 200 ng/ml human Fas monoclonal antibody (clone CH-11) for 9 hr. Genomic
DNA was isolated from uninduced and induced Jurkat cells. The LM-PCR assay was performed according to the User Manual (15 PCR cycles). 15 l of each reaction was electrophoresed on a 1.2% agarose/EtBr gel. Lane 1: uninduced. Lane 2: induced (apoptotic).
58
-OH
www.bdbiosciences.com
Product Listing
Description
Apps.
Format
Size
Cat. No.
Kit
FA
50 rxns
K2021-1
Notice to Purchaser
These products are optimized for use in the Polymerase Chain Reaction
(PCR) covered by patents owned by Hoffmann-La Roche and F.
Hoffmann-La Roche Ltd. Under these patents no license to use the PCR
process is conveyed expressly or by implication to the purchaser by the
purchase of these products. A license to use the PCR process for certain research and development activities accompanies the purchase of
certain reagents from licensed suppliers, such as CLONTECH
Laboratories, Inc., when used in conjunction with an authorized thermal cycler, or is available from Perkin-Elmer Corporation. Further information on purchasing licenses to practice the PCR process may be
obtained by contacting the Director of Licensing at the Perkin-Elmer
Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404 or Roche
Molecular Systems, Inc., 1145 Atlantic Avenue, Alameda, CA 94501.
References
1. Wyllie AH. 1980. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature.284(5756):555-6.
2. Arends MJ, Morris RG, Wyllie AH. 1990. Apoptosis. The role of the endonuclease. Am J Pathol. 136(3):593-608.
3. Staley, K., Blaschke, A. J., and Chun, J. 1997. Apoptotic DNA fragmentation is detected by a semiquantitative ligation-mediated PCR of blunt DNA ends. Cell Death Diff. 4:66-75
The ApoAlert LM-PCR Ladder Assay Kit was cited in the following articles:
Yasunori Kasahara, Rubin M. Tuder, Laimute Taraseviciene-Stewart, Timothy D. Le Cras, Steven Abman, Peter K. Hirth, Johannes Waltenberger, and Norbert F. Voelkel. 2000. Inhibition of VEGF
receptors causes lung cell apoptosis and emphysema. J. Clin. Invest. 106(11):13111319
Eric Tse and Terence H. Rabbitts. 2000. Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy. Proc. Natl. Acad. Sci. 97(22):12266-12271
BD Clontech
www.bdbiosciences.com
59
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