Gel Electrophoresis

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 Transfer
Membranes

“Thinking without knowledge

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makes chance the ruler.”

www.
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.com

www.

Werner Kollath, German bacteriologist

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Detergents Kontaminationen Immunoassay Gel Electrophoresis

Nukleinsäuren
Probleme & praktische Lösungen

durch
Buffer Size Marker
Ever since the foundation of the firm, AppliChem has

invested extensively in communication and marketing. The

Take the Pink Link!

fresh and unusual appearance attracted great attention in
Take the Pink Link!

www. Take the Pink Link!

.com
www.
.com Take the Pink Link!

the market from the very beginning. Our growth confirms

www.
.com
www.
.com

Agarose-
Elektrophorese the relevance of these measures. Wichtiges
Wissenswertes
Wunderbares
aus Chemie & Biologie
AppliCations Nr.1
AppliCa
We offer our customers an extensive library, with numerous
Improving quality o
Nukleinsäure-Dekontamina
mit der ExitusPlus™-Tec tion
hnologie
Die moderne Gentechnik
zeigt, dass in vielen Fällen
schon freie DNA-Moleküle
für Infektionen, Rekombin Using ready-to-use ELISA

brochures and applications whose use makes everyday life

ationen oder biologische kits from manufactu
Transformationen ausreiche
[1,2]. Zusätzlich werden n
die Nachweisverfahren times however, home-ma
für DNA-Moleküle immer de ELISA is required b
sensitiver. Daher wird die
Detektion von Kontaminationen the right antibodies or the
oder die Verhinderung characteristics of the
von Amplifikations-Artefakten
in der PCR für die Gentechn detection are not appropria
ik, die Kriminalistik, die te. Ready-to-use EL
Biomedizin und die Hygiene
immer wichtiger. Die vollständi stored for two years at 4°C
ge Dekontamination without any probl
von Geräten und Materialie
n von DNA-Molekülen wird
so zu einem entscheidenden completely different story.
Faktor für die allgemeine For any new measur

in the laboratory easier.

biologische Sicherheit.
because after storage of some
days the plates d

Take the Pink Link! Keywords Alles oder Nichts: Erstaunlich

e Erkenntnis
Das Mittel der Wahl zur Beseitigung von
Nukleinsäure- Kontaminationen durch Nukleinsäuren
Kontamina Keywords
Take the Pink Link!
– ein Mittel das alles zerstört, nicht nur ist immer noch Chlorbleichlauge („bleach“) Why is there such a great difference
Dekontamination Köln, nach einer unschädlichen Alternative
die Nukleinsäure. Dies hat uns veranlasst
in Kooperation mit multiBIND Biotech, The reason is that in professional ELISA
in storage between h
.com zu suchen und die molekulare Wirkungsweise Immunoassays kit production the plates are not
DNA-Degradationstest sonstigen DNA-Dekontaminationsmittel der auf dem Markt befindlichen easy to perform process has been an industry

A comprehensive catalogue of products, with detailed

www. zu untersuchen.
suchen. Hierfür wurde unter sehr hoher standard for thirty years. Fo
.com schuss) mit definierten DNA-Kontami
nationen die Eigenschaften der konventionelle Belastung (großer DNA-Über
DNA-Über- Antibody Stabilisation coating stabiliser solution. It is just as
simple as a “second blocking step
PCR-Test werden offensichtlich: Erstens werden n Mittel verglichen. Zwei Probleme lutions freely available in low volumes for
durch die konventionellen Mittel in keinem ELISA Plates use in research lab until now. AppliC
und zweitens enthalten diese Mittel Komponenten Fall die DNA-Moleküle effizient zerstört in volumes starting as small as 50 ml, which
Autoklavieren von DNA mit stark korrosiven oder giftigen Eigenschaften is called the AppliCoat Plate Stabi
für uns die Notwendigkeit der Neuentwicklun . Als Fazit daraus hat sich to-use and has a great advantage compared
g einer effektiven Lösung zur DNA-Dekontam Cross-reactivity to almost any stabiliser used
DNA-ExitusPlus™ und Autoclave-Exit ination ergeben, die wir hier als antibodies and antigens than most other
usPlus™ vorstellen. Im Vergleich zu products do. And there is a secon
RNA schnell und effizient zerstört, ohne den herkömmlichen Produkten wird Interfering effects
dass das Reagenz korrosive oder giftige DNA und
Bei der DNA-Dekontamination unterscheidet Eigenschaften aufweist. Two benefits with one solution
man nach der molekularen Wirkungsweise
prinzipien zur Zerstörung oder Inaktivierung der eingesetzten Mittel drei Grund- When antibodies are coated onto ELISA
der genetischen Information: Modifika
Modifikation, Denaturierung und Degradation. plates, most of the antibodies are not
Je nach Zusammensetzung der Mittel into close contact to the plastics surface
können diese drei Prinzipien einzeln of the ELISA plate, conformational ch
Da nach den aktuellen Erkenntnissen oder in Kombination angewandt werden. The result is that most antibodies coated

information provided by no other catalogue in the field,

zum biologischen Risikopotenzial von on a plate are unfolded or inactive. O
DNA-Dekontamination die Zerlegung freien DNA-Molekülen für eine wirklich sichere active and can bind to analytes and this
dieser DNA-Moleküle in möglichst kleine is greatly variable depending on the s
wurden die gängigen konventionelle Fragmente die wirkungsvollste Methode really differ from batch to batch or even
tionellenn Mittel mit unserer Neuentwicklun ist, from well to well.
glichen. Der DNA-Degradationstest erlaubt g DNA-ExitusPlus™ im DNA-Degradat These differences from well to well can
einen sensitiven, quantitativen Vergleich ionstest ver
ver- affect the variability of an assay, bec
(Abb. 1 und 2). der Geschwindigkeit des DNA-Abbaus way of refolding antibodies and of preserving
antibodies from conformati
Unerwarteter Weise haben wir festgestellt, decrease such variabilities in assay performance.
dass einige der bekannten kommerziellen This is a key benefit of Ap
kation oder Denaturierung der DNA-Moleküle Mittel nur mit dem Prinzip der Modifi- coated proteins to refold and then to
arbeiten. Eine Zerlegung der DNA-Stränge preserve active conformation over a lo
genetische Informa
Information, für die diese DNA-Stränge erfolgt dabei nicht, sondern die antibody conformation of some of the
kodieren, wird eigentlich nur maskiert. coated antibodies and 2. Preserving c
der DNA-Moleküle durch Entfernung Eine chemische Demaskierung fits are used for production of high-quality
der blockierenden Gruppen würde die ELISA kits as well as in resear
plifizierbar machen. Nach dem heutigen genetische Information wieder lesbar Stabiliser the percentage of active antibodies
Wissensstand zur Gentechnik und der und am- will still be in the range of 2–8
trägern sind solche Mittel eigentlich nicht Problematik der Neukombination von from well to well and from plate to plate

is available in German and English.

mehr zeitgemäß. Aber auch die Mittel, Erb- can be minimised in most assays
die zu einer nachweisbaren Degradation depend on the used antibodies, but when
ELISA are validated (e.g. according
Validation”, FDA, 2001) or according to
other validation strategies, the differe
The positive effects of AppliCoat Plate
Stabiliser are shown in Fig. 1. A sandwi

AppliCations AppliChem brings advantages through knowledge.

Nr.5
AppliCations AppliCations
Dekontamination der Haut No.6
und Hände von Nukleinsäure
n Size-Exclusion Chromato
graphy
Nr.3
for purification of biomolec DNA-freie Reagenzien
ules
Freie Nukleinsäuren verursach
en als Kontaminationen
große Probleme im
und Mastermixe für die PCR
Forschungs- und molekular Size-exclusion chromatog
biologisch-analytischen oder raphy (SEC) is a popular method
klinisch-diagnostischen to separate biomolecules
Labor. Durch die extrem hohe based on their size. Primarily,
Sensitivität von DNA-Nach it is applied to the separation Der Anteil von molekular
weistests, können kleinste of biopolymers such as biologischen Nachweismethoden
Verunreinigungen in PCR-Ansä proteins and nucleic acids, ist in den letzten Jahren
tzen zusätzliche Arbeit bedeuten i.e. water-soluble polymers.
und im schlimm- This system is also called erheblich gestiegen, besonder
filtration, typically with beads gel s in den Bereichen Qualitätsk
sten Fall Ergebnisse verfälsch of dextran or agarose serving ontrolle, Forensik,
en. Mit Derma-ExitusPlus™ as gel matrix. Smaller klinischer Forschung und
(HHDK) aus der Serie molecules pass significant Diagnostik – insbesondere
von ExitusPlus™-Produkten ly slower through the column der Infektionsdiagnostik
wird erstmals ein völlig than larger molecules. Not .
neuer Anwendungsbereich be mixed up with gel electropho to Gerade für diese Applikatio
resis, there are big difference nen werden hochsensitive
erschlossen bzw. zusätzlich
e Kontaminationsquellen
ausgeschlossen. Size Marker • © 2010 AppliChem separation principle. SEC
not separate small molecules
does not require electric current
s in terms of the
and the sieving effect will
PCR-Tests benötigt. Dafür
und gleichzeitig zuverläss
bietet AppliChem nun optimiert
e PCR-Kits an und widmet
ige

first. sich explizit der Hintergru

ndproblematik, die durch
DNA belastete Reagenzie
n und
Arbeitsplätze entstehen kann.

ine der Hauptquellen für Kontamination

en mit Nukleinsäuren ist der Experimentato
B. aus Hautschuppen, Haaren und Speichel r selbst. Die Nukleinsäuren stammen
oder von Mikroorganismen, die seine
eigesetzt werden. Gelangen diese in Haut besiedeln oder z.B. beim Niesen Keywords It is indeed correct that smaller molecules
die PCR-Ansätze oder PCR-Reagenzi pass more slowly through t
en, können s
contents
1 DNA marker 2
1.1 Tips on the use of DNA length markers 2

1.2 AppliChem DNA marker overview 4

1.3 DNA marker 5

2 Dyes for nucleic acids 16

2.1 Overview 16

2.2 Methylene blue 16

2.3 Ethidium bromide 18

3 Protein marker 19
3.1 AppliChem protein marker overview 19

3.2 Precision protein markers 19

3.3 Prestained protein markers 21

4 Dyes for protein gels 24

4.1 Overview 24

4.2 Coomassie stain 24

4.3 Proteo-Dye 26

© 2010 AppliChem • Size Marker 1

 Tips on the use of DNA length markers
Correct storage

1.1
Deproteinised and lyophilised DNA samples are extremely stable (> 5 years). Problems do not usually occur unless DNA
markers in solution are stored (> 6 weeks) at room temperature, they become contaminated with bacteria, or are frequent-
ly ­thawed and refrozen (> 20 times). After dissolution of the DNA, we therefore recommend aliquoting of the DNA marker
in ready-to-use aliquots for storage at -20°C (for 2-4 years). At +4°C, markers in solution are stable for several weeks
or even months. Here, however, there is the risk of bacterial or nuclease contamination. This can be prevented by storage
at -20°C.

End-labeling
Thanks to their deproteinised and lyophilised form, these DNA markers are suitable for universal use for end-labeling with
modified nucleotides. Labeling in four positions via the terminal EcoR I generated recessed ends is possible, especially with
the DNA ­ladder 100 bp (A3470) and the DNA Ladder Mix 100-5000 (A3660). For the labeling of the DNA, the product is
simply dissolved in TE buffer or bidistilled water.

DNA staining with methylene blue

Occasionally, ethidium bromide is not suitable for staining DNA. An alternative is methylene blue. In such cases, however, it
must be kept in mind that the mass of DNA used must be increased by about 30 % and that about 1.5 h more must be ­planned
for destaining steps. With a methylene blue concentrate supplied by AppliChem, you achieve very good results, even for small
fragments of about 200 bp.

Mass calculations for single fragments

Using DNA markers of a defined origin, the calculation of the mass of single fragments is relatively easy. The amount loaded
per lane, e.g. 1 µg/10 µl, is divided by the number of base pairs of the DNA used and is multiplied by the fragment size. For
example: the 267 bp fragment of the pBR322 Hae III marker with a loading amount of 1 µg: 1 µg : 4361 bp (pBR322) =
0.229 ng/base pair x 267 bp of the fragment = 61.2 ng/267 fragment.
Assuming comparable staining (saturation with dye), the mass of unknown fragments can be determined in this way. If
­required, concentration gradients can be loaded.

Less is often more

The different gel formats for agarose and polyacrylamide gel electrophoresis and the varying sensitivity of staining or detec-
tion mean that it is only possible to give an approximation of the recommended DNA amount to be loaded. Most DNA mar-
kers show the best separation with loading amounts of 0.5-1 µg on ­agarose gels. The general rule is: the lower the number
of marker bands, the smaller the total amount needed. A low loading amount is also of advantage with short migration
distances or very ­sensitive detection. When using polyacrylamide gels, generally only about 1/5-1/2 the amount required for
agarose gels is necessary. For DNA markers with large fragments and a high mass (e.g. equimolar mixtures or lambda DNA
markers), depending on the agarose concentration, a minimum separation distance is required to separate or completely
distinguish between the dominant upper bands in terms of width. This separation distance can often be reduced by 10-15
% by ­reducing the marker amount. By using equalized markers, in addition to a marked reduction of the marker amount,
the ­separation distance can be reduced by a further 10-15 %, from e.g. 70 to 50 mm, on a standard format gel, to obtain
the best results.

i n t r o
2 Size Marker • © 2010 AppliChem
How to achieve results quickly
Especially deproteinised DNA length markers can be separated very well using high voltages, e.g. within 20 minutes with a
migration distance of 70 mm on a 1.2 % agarose gel at 120 Volt. This can only be realized, however, with a high quality gel
­migration buffer (the water quality must be taken into account!) and an adequate amount of buffer (400-600 ml). Poor
buffer quality results in overheating of the agarose gel at higher voltages and this therefore causes problems. A high buffer
volume (the buffer can usually be reused 4-6 times in one week without problems) leads the heat off, provided the gel
chamber for submersion is of adequate size.

Staining front too intense?

In some cases, users who regularly perform assays with our products sometimes comment that the dye concentration in the
gel loading buffer we supply is too high (only lyophilised markers). If this is the case, simply dissolve the DNA length ­marker
in double concentration in the gel loading buffer and further dilute it with 1/2 volume TE buffer. Even when using a 50 %
solution, there are no problems with the resulting 7 % glycerol concentration to increase the solution density.

Plausibility of the results and ion concentrations:

Non-plausible results (e.g. unexpected fragment runs at 500 bp instead of 350 bp) may occur under relatively extreme
­conditions (separation of restriction or PCR samples with a very high salt content > 150 mM) or at very high voltages with
relatively short separation distances. To identify errors, 1/10 volume restriction buffer can be added to the aliquot of the DNA
marker, for example, or the restriction sample can be appropriately diluted with gel loading buffer (usually 1/2 volume). In
many cases, lowering the voltage during separation is also helpful. The accurate determination of the size of fragments or
PCR ­products may also be impaired, when salt fronts combined with local problems of leading off heat with conducting away
heat and very high ­electrophoresis voltages, result in partial denaturation of the DNA fragments. These are conditions that
theoretically may also occur when processing samples from Maxam-Gilbert sequencing in combination with sample buffers
containing formamide.

d u c t i o n
© 2010 AppliChem • Size Marker 3
 Prod. No. DNA Marker Bands Fragment Sizes [bp]
A3470 DNA Ladder 100 bp (lyophilised) 11 1000 900 800 700 600 500 400
A5191 DNA Ladder 100 bp 10 1000 900 800 700 600 500 400
A3302 DNA Ladder 100 bp equalized (lyophilised) 11 1000 900 800 700 600 500 400
A5216 DNA Ladder 100 bp plus 11 1500 1000 900 800 700 600 500
A3660 DNA Ladder Mix 100-5000 (lyophilised) 17 5000 4000 3000 2500 2000 1500 1000
A3982 DNA Ladder 250 bp (lyophilised) 16 8000 6000 5000 4000 3000 2750 2500
A8640 DNA Ladder 250 bp plus (lyophilised) 15 12000 10000 8000 6000 5000 4000 3000
A2667 DNA Ladder 1 kb (lyophilised) 11 10000 8000 6000 5000 4000 3000 2500
A5207 DNA Ladder 1 kb 13 10000 8000 6000 5000 4000 3000 2500
A6430 DNA Ladder 1 kb concatamer (lyophlised) >25 1000 bp steps (1000-approx. 25000)
A7215 DNA Marker quick-run (lyophylised) 5 2500 2000 1500 1000 500
A7222 DNA Marker quick-run extended (lyophylised) 9 6000 4000 25000 2000 1500 1000 500
A4406 DNA Marker pBR322 - Hae III (lyophilised) 22 587 540 502 458 434 267 234
A5229 DNA Marker pBR322 - Hae III 22 587 540 502 458 434 267 234
A6927 DNA Marker pBR328 Mix (lyophilised) 12 2176 1766 1230 1033 653 517 453
A5194 DNA Marker Phage Lambda Sty I 11 19329 7743 6223 4254 3472 2690 1882
A4412 DNA Marker Phage Lambda BstE II (lyophilised)
BstE II (lyophilised)
Bst 14 8454 7242 6369 5686 4822 4324 3675
A5220 DNA Marker Phage Lambda BstE II
BstE II
Bst 14 8454 7242 6369 5686 4822 4324 3675
A5589 DNA Marker Phage Lambda Hind III (lyophilised)
Hind III (lyophilised)
Hind 8 23130 9416 6557 4361 2322 2027 564
A5223 DNA Marker Phage Lambda Hind III
Hind III
Hind 8 23130 9400 6557 4361 2322 2027 564
A5235 DNA Marker pUC19 - Msp I 12 501 489 404 331 242 190 147
A3996 DNA Marker pUC19 - Msp I (lyophilised) 12 501 489 404 331 242 190 147

Lyophilised markers
Starting material
The DNA in our lyophilised length markers is amplified in low-nuclease host bacteria, restricted with quality enzymes, and
is deproteinised. This ensures that they contain high-quality, low-protein or nuclease-free products with very good migration
properties on agarose, agar and polyacrylamide gels.

Quality control
Numerous gel separations of undigested plasmids and digested fragments form part of the manufacturing process. The
fragment mass is determined using two calibrated photometers via conversion (1 OD260 nm = 50 µg dsDNA) and is aliquoted
at 100 % (+2 %). Each batch of DNA and gel loading buffer is subject to a final quality check using gel electrophoresis.

Stability
Lyophilisation of the DNA markers results in very stable products, without any significant impairment of quality at
room temperature after transport (even at the height of summer, > 35°C for several days) or after long storage times
(> 4 years at -20°C).

Resuspension
Before use, lyophilised DNA markers can be dissolved in gel loading buffer or optional for end-labeling in sterile, bidistilled
water. The lyophilised form allows you the highest degree of flexibility (labeling, concentration, intensity of staining
bands).

Legal note
The DNA ladders were manufactured by digestion of plasmids with EcoR I. The plasmids are legally protected. To produce
copies apply for a permission from the manufacturer.

4 Size Marker • © 2010 AppliChem

 1.2
300 200 150 100
300 200 100
300 200 150 100
400 300 200 100
900 800 700 600 500 400 300 200 150 100
2250 2000 1750 1500 1250 1000 750 500 250
2000 1750 1500 1250 1000 750 500 250
2000 1500 1000 500
2000 1500 1000 750 500 250

200 100
213 192 184 124 123 104 89 80 64 57 51 21 18 11 8
213 192 184 124 123 104 89 80 64 57 51 21 18 11 8
394 298 234 220 154
1489 925 421 74

selection guide
2323 1929 1371 1264 702 224 117
2323 1929 1371 1264 702 224 117
125
125
111 110 67 34 26
111 110 67 34 26

DNA Ladder 100 bp (lyophilised)

DNA marker
DNA size standard for medium-sized fragments and PCR products
A3470

Description The fragments of this size marker occur in equimolar proportions, i.e. also all possible marking
positions on the terminal EcoR I generated recessed ends. The band mass increases with fragment

1.3
length. The DNA is deproteinized and lyophilized. This marker is particularly suitable for com-
parisons with medium-sized DNA fragments and PCR products. The 500 base pair band mass has
been doubled for easier orientation on 1.5-2.0 agarose gels. The size and mass of plasmid in - ser-
tions or PCR products can be determined precisely in the range of up to 1000 base pairs at
intervals of 100 base pairs. An additional band with 150 base pairs has been included for the
deter mination of smaller PCR products. The best results are achieved after a migration distance of
approx. 70-80 mm on agarose gels. Each lane of a normal sized agarose gel (approx. 80 ml)
should be loaded with approx. 0.4-0.8 µg of marker. Loading buffer is supplied separately.
Number of bands 11
Fragment sizes (bp) 1000, 900, 800, 700, 600, 500 (x2), 400, 300, 200, 150, 100
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Storage -20°C
Loading volume 0.4-0.8 µg per lane
Package size A3470,0050 50 µg

© 2010 AppliChem • Size Marker 5

 The quality of lyophilised DNA sizing standards differs considerably from products offered by most other manu­
facturers thanks to the elaborate manufacturing method, which also fulfils the highest ­quality requirements!
The digested DNA is not simply precipitated and resuspended, but also extracted in phenol. This guarantees the
extremely long shelf-life without any loss of quality. Not only the prices should therefore be compared.

DNA Ladder 100 bp A5191

Description 1 00 bp DNA ladder is ideal for determining the size of double-stranded DNA from 100 to 1 000
base pairs. The 100 bp and 500 bp fragments are present at increased intensity to allow easy
identification. All fragments are blunt-ended.
Number of bands 10
Fragment sizes (bp) 1000, 900, 800, 700, 600, 500 x 2, 400, 300, 200, 100 x 2
Storage buffer 10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
Concentration 0.2 mg/ml
Storage -20°C
Loading volume 1 µg per lane
Package sizes A5191,0005 0.05 mg (= 250 µl)
A5191,0025 0.25 mg (= 1.25 ml)
Assay conditions:
1.7 % agarose

DNA Ladder 100 bp equalized (lyophilised) A3302

DNA size standard for medium-sized fragments and PCR products
Description With this marker, the band mass of the larger fragments have been reduced and have
been reinforced for the smaller fragments (as compared to marker A3470) by up to
a factor or 4.5. The result is an even appearance in intensity of the individual bands.
The mol number of the fragments decreases from the larger to the smaller fragments,
as do the possible marking positions on the terminal EcoR I generated recessed ends.
The 500 bp band, with an increase in mass of a ­factor of 3, enables rapid orientation on
1.5-2.0 % agarose gels. Thanks to the even distribution, a shorter migration distance is
needed for the larger fragments, in order to achieve a clear ­distinction between the bands.
The best results are achieved after a migration distance of approx. 60-80 mm on agarose
gels. Each lane of a normal sized ­agarose gel (approx. 80 ml) should be loaded with approx.
0.2-0.5 µg of marker. The ­equalized 100 bp ladder is ­therefore very economical in use.
Loading buffer is supplied separately.
Number of bands 11
Fragment sizes (bp) 1000, 900, 800, 700, 600, 500 (x3), 400, 300, 200, 150, 100
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA;
loading buffer (1X) 10 % glycerol; 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.2-0.5 µg per lane
Storage -20°C
Package size A3302,0020 20 µg

DNA marker
6 Size Marker • © 2010 AppliChem
DNA Ladder 100 bp plus A5216

Description 100 bp + 1.5 kb DNA Ladder is suitable for sizing linear double-
s­ tranded DNA fragments from 0.1 to 1.5 kb. Use in combination with a
loading buffer (please see our present catalog for a list of loading
buffers).
Number of bands 11
Fragment sizes (bp) 1500, 1000 (x2), 900, 800, 700, 600, 500 (x2), 400, 300, 200, 100 (x2).
The 500 bp and 1 kb bands are brighter than the other bands in the ladder.
Concentration The marker is supplied in a concentration of 0.2 mg/ml
in 10 mM Tris · HCl (pH 8.0), 1 mM EDTA, 10 mM NaCl.
Storage Store at -20°C. Prepare small aliquots to prevent repeated
freeze & thawing!
Loading We recommend loading of 0.4-0.6 µg (2-3 µl) per lane.
Package sizes A5216,0005 0.05 mg (= 250 µl)
A5216,0025 0.25 mg (= 1.25 ml) Assay conditions:
1.7 % agarose

DNA Ladder Mix 100-5000 (lyophilised) A3660

DNA size standard for the determination of the size of medium-sized fragments and plasmids
Description The 100 bp ladder (A3470) was extended with fragments in the size range
of 1500-5000. The extension by 40 % mass in the larger fragments means easy
­orientation from 1500 bp upwards on 1.2-1.5 % agarose gels. In addition to small and
large plasmid insertions and PCR products, the size and quantity of plasmid­ ­vectors
can also be determined. The best results are achieved after a migration distance
of 80-90 mm on agarose gels with loading volumes of 0.5-0.8 µg per lane on
standard sized gels.
Number of bands 17
Fragment sizes (bp) 5000, 4000, 3000, 2500, 2000, 1500, 1000, 900, 800, 700, 600,
500 (x2), 400, 300, 200, 150, 100.
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA;
loading buffer (1X) 10 % glycerol; 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.5-0.8 µg per lane (for good staining properties with ethidium bromide)
Migration distance Best results are achieved after a migration distance of 70-90 mm.
Storage -20°C. Avoid repeated thawing and refreezing. Portioning in small aliquots is
recommended. The lyophilised product is stable for many weeks even at room
temperature!
Package size A3660,0050 50 µg

This sizing standard was manufactured using digestion of 7 plasmids with EcoR I. The plasmids are legally
p­ rotected. Permission to produce copies must be sought from the manufacturer. The DNA was deproteinised,
­precipitated and lyophilised after digestion with phenol/chloroform. If the ­fragments are to be labeled, the­ marker
should be dissolved in distilled water (10 minutes at room temperature, shaking occasionally).

© 2010 AppliChem • Size Marker 7

 DNA Ladder 250 bp (lyophilised)  A3982
Universal DNA size standard for the determination of the size of plasmids and their DNA insertions
Description With its two clearly reinforced bands at 2500 and 1500 base pairs, the 250 bp ladder enables
rapid orientation on 1.0-1.2 % agarose gels. Using gel electrophoresis, with intervals of 250 base
pairs in a range of 250-3000 base pairs, it is possible after restriction to determine clearly and
precisely, for example, large plasmid insertions, and vector plasmids above 3000 base pairs with
intervals of 1-2 kb. In comparison to DNA markers with equimolar band distribution, the mass of
the smaller bands of this 250 bp ladder up to 1000 bp has been reinforced and the larger bands
above 3000 bp have been reduced. This means that optimum results are already achieved after
a migration distance of 60-80 mm on agarose gels and that small amounts of about 0.7 µg per
lane can be used with standard sized agarose gels (80 ml).
Number of bands 16
Fragment sizes (bp) 8000, 6000, 5000, 4000, 3000, 2750, 2500 (x3), 2250, 2000, 1750, 1500 (x3), 1250, 1000, 750,
500, 250
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.4-0.8 µg per lane (for good staining properties with ethidium bromide)
Migration distance Best results are achieved after a migration distance of 75-85 mm.
Storage -20°C. Avoid repeated thawing and refreezing. Portioning in small aliquots is recommended. The
lyophilised product is stable for many weeks even at room temperature!
Package size A3982,0050 50 µg

This sizing standard was manufactured using digestion of plasmids with EcoR I. The plasmids are legally protected.
Permission to produce copies must be sought from the manufacturer. The DNA was deproteinised, precipitated and lyophilised
after digestion with phenol/chloroform. If the fragments are to be labeled, the marker should be dissolved in distilled water
(10 minutes at room temperature, shaking occasionally).

DNA Ladder 250 bp plus (lyophilised)  A8640

DNA size standard for genomic DNA, plasmids and their insertions
Description The DNA Ladder 250 bp plus is intended for use in the sizing of DNA fragments during
cloning and of genomic DNA. By combining two groups of fragments differing in the
fragment size range, smaller fragments (250-2000 bp) as well as plasmid vectors and
fragment of genomic DNA (3000-12000 bp) my be sized on the same gel. The mass
of the larger fragments is reduced to achieve a better separation on short distances of
70-90 mm only. The terminal Eco RI restriction sites allow for an efficient labeling of
the fragments. Best results are achieved by loading 0.8-1.0 µg/lane on a 1.2 % agarose
gel (standard minigel size). The DNA is supplied deproteinized and lyophilized.
Number of fragments 15 bands
Fragments sizes (bp) 12000, 10000, 8000, 6000, 5000, 4000, 3000, 2000, 1750, 1500, 1250, 1000, 750,
500, 250
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.4-0.8 µg/lane (e.g. 1.0-1.2 % agarose gel)
Storage Store at -20°C. Store as small aliquots, if resuspended.
Package size A8640,0050 50 µg

8 Size Marker • © 2010 AppliChem

DNA Ladder 1 kb (lyophilised) A2667

Description The size of medium-sized plasmids and fragments can be determined with 11 DNA bands
between 10,000 and 500 base pairs. Unlike the equimolar distribution of the bands in other
DNA markers and the long migration distance necessary with these for the separation
of large ­fragments, the mass of the larger bands of the 1 kbp DNA ladder has been
reduced. This permits relatively short separation distances for a 1 kbp DNA ladder
(approx. 70 mm on 1.2 % agarose gels) and means that it is very economical in use
(0.4-0.6 µg per lane, or 400 separations) on normal sized gels. Loading buffer is
supplied separately. The fragments have terminal EcoR I generated recessed ends.
The DNA is deproteinized and lyophilized.
Number of bands 11
Fragment sizes (bp): 10000, 8000, 6000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 500
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.4-0.6 µg per lane
Storage -20°C
Package sizes A2667,0050 50 µg
A2667,0200 4 x 50 µg

DNA marker
DNA Ladder 1 kb A5207

Description 1 kb DNA Ladder is a convenient marker for determining the size of

d ouble-stranded DNA from 250 to 10,000 base pairs. The 1,000 and
3,000 bp fragments have increased intensity relative to the other bands
on ethidium bromide-stained agarose gels, and serve as reference
­indicators. All fragments are blunt-ended.
Number of bands 13
Fragment sizes (kb) 10.0, 8.0, 6.0, 5.0, 4.0, 3.0 (x2), 2.5, 2.0, 1.5, 1.0 (x2), 0.75,
0.5, 0.25
Storage buffer 10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
Concentration 0.2 mg/ml
Storage -20°C
Package sizes A5207,0005 0.05 mg (= 250 µl)
A5207,0025 0.25 mg (= 1.25 ml)

© 2010 AppliChem • Size Marker 9

 DNA Ladder 1 kb concatamer (lyophilised) A6430
DNA size marker as alternative to irregular l-fragments (from 1 to approx. 25 kb)
Description A partial ligation of 1000 bp Hind III fragments results in a DNA ladder ranging from 1000 to
approx. 22000 bp in 0.8 to 1% gels using standard agarose (Prod.-No. A2114; separation
range up to approx. 25 kbp). The upper limit of the fragment ligation results in the main
mass of the ligation products in the separation range optimal for standard agaroses (agar).
For a better orientation, the 3000 bp and 10,000 bp bands are brighter. Optimal results will be achieved
after a distance of approximately 80-100 mm in agarose gels and a loading volume of approximately
1 µg per lane in normal-sized gels. This 1000 bp concatamer ladder may be applied either in reduced
loading volumes (for a seperation range e.g. < 10000 bp) or in increased volumes (> 15000 bp).
Even under optimized ligation conditions, an intramolecular religation may occur, especially with
smaller fragments. Therefore, at high gel loading volumes, ligation products (closed circles) have been
observed between the 1000 and 3000 bp bands. For a better orientation, the 3000 bp band is brighter.
Number of bands > 25
Fragment sizes (bp) 1 000 bp steps (1000 - approx. 25000)
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume approx. 1.0 µg per lane
Migration distance Optimum results after a distance of 15-25 mm only.
Storage -20°C.
Package size A6430,0050 50 µg

This marker has been designed as an alternative for irregular λDNA fragment markers, for the separation in standard low endoosmosis agarose
(Prod.-No. A2114; separation range 70 bp to approximately 25 kbp). In comparison to other concatamer markers, it has a reduced mass in the
non-separating range (high molecular weight range), resulting in a better resolution (good readings up to 24 kbp). For applications requiring
a good separation in the high molecular weight range pulsed-field electrophoresis is recommended. Due to the production procedure (ligation
of 1000 bp Hind III fragments), it is impossible to determine the specific mass of a single band. The DNA of this marker (50 µg sufficient for
approximately 100 loadings) is deproteinised, Iyophilised and is supplied with 1 ml of the 1 x gel loading buffer.

DNA Marker quick-run (lyophilised)  A7215

DNA size standard for determination of DNA fragments after short gel runs (15 - 25 mm)
Description This marker is ideal for use with mini gels, since its bands are separated very well even
after very short gel runs as short as 15-25 mm in an e. g. 1.5-1.8 % agarose gel. The
single bands have been equalized in terms of their mass. The 1500 bp fragment has
an increased mass for better orientation. Optimum results are achieved in a 1.5-1.8
% agarose gel with as little as 0.25 µg of the marker per lane (normal sized mini gel).
Please note that the DNA fragments have to be saturated with ethidium bromide in
the running buffer during the separation.
Number of bands 5
Fragment sizes (bp) 2500, 2000, 1500 *, 1000, 500
The mass of the marked fragment* has been increased for better orientation.
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.015 % bromophenol blue
Loading volume 0.25 µg per lane (for a good detection with ethidium bromide)
Migration distance Optimum results after a distance of 15-25 mm only.
Storage -20°C. Prevent repeated freezing and thawing (> 20x). We recommend to prepare
small aliquots. The lyophilised form is stable even for weeks at ambient temperature
Package size A7215,0050 50 µg

This marker is made of plasmids with specific mutagenesis sites. The Eco RI-digested DNA has been deproteinized with phenol/chloroform,
desalted, precipitated and lyophilised. Resuspend the DNA in the sterile-filtered gel loading buffer (supplied with the marker) by incubation for
10 minutes at room temperature with occasional shaking.

10 Size Marker • © 2010 AppliChem

DNA Marker quick-run extended (lyophilised) A7222
DNA size standard for determination of DNA fragments after short gel runs (35 mm)
Description This marker is ideal for use with mini or midi gels, since its bands are separated
very well even after very short gel runs as short as 35 mm in an e. g. 1.2 % agarose
gel. The single bands have been equalized in terms of their mass. The 1500 bp
fragment has an increased mass for better orientation. Optimum results are achieved
with as little as 0.3-0.5 µg of the marker per lane (normal-sized mini/midi gel).
Number of bands 9
Fragment sizes (bp) 6000, 4000, 2500, 2000, 1500*, 1000, 500, 200, 100
The mass of the marked fragment* has been increased for better orientation.
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.015 % bromophenol blue
Loading volume 0.3 - 0.5 µg per lane
(for a good detection with ethidium bromide in a mini or midi gel)
Storage -20°C. Prevent repeated freezing and thawing (> 20x). We recommend to prepare
small aliquots. The lyophilised form is stable even for weeks at ambient temperature!
Package size A7222,0050 50 µg

This marker is made of plasmids with specific mutagenesis sites. The Eco RI-digested DNA has been deproteini-
zed with phenol/chloroform, desalted, precipitated and lyophilised. Resuspend the DNA in the sterile-filtered gel
loading buffer (supplied with the marker) by incubation for 10 minutes at room temperature with occasional
shaking.

DNA Marker pBR322 - Hae III (lyophilised)

DNA marker A4406
DNA size standard for high-percentage agarose gels and polyacrylamide gels
Description Plasmid pBR322 was digested with Hae III, deproteinized and lyophilized. This
marker is particularly suited to comparisons with small PCR fragments and DNA
from restriction samples on polyacrylamide gels or high-percentage agarose gels
(> 2.2 %). The best results are achieved after a migration distance of approx.
70-80 mm on agarose gels or 100 mm on 6-8 % polyacrylamide gels. Each lane of
a normal sized gel (approx. 80 ml) should be loaded with approx. 1 µg of ­marker
for agarose gels or 0.5 µg for PAA gels. Loading buffer is supplied separately.
Number of bands 22
Fragment sizes (bp) 587, 540, 502, 458, 434, 267, 234, 213, 192, 184, 124, 123, 104, 89, 80, 64, 57,
51, 21, 18, 11, 8
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.5-1.0 µg per lane
Storage -20°C
Package size A4406,0050 50 µg
Please note! Extremely small fragments can only be visualized on high-percentage PAA gels.

© 2010 AppliChem • Size Marker 11

 DNA Marker pBR322 - Hae III A5229

Description This marker is generated by digestion of the plasmid pBR322 with Hae III.
Number of bands 22
Fragment sizes (bp) 587, 540, 502, 458, 434, 267, 234, 213, 192, 184, 124, 123, 104, 89, 80, 64, 57,
51, 21, 18, 11, 8
Storage buffer 10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
Concentration 0.2 mg/ml
Storage -20°C
Package sizes A5229,0005 0.05 mg (= 250 µL)
A5229,0025 0.25 mg (= 1.25 ml)

Assay conditions:
1.7 % agarose

DNA Marker pBR328 Mix (lyophilised) A6927

DNA size standard for middle-sized fragments and PCR products
Description Plasmid pBR328 was digested with Hinf I and Bgl I, respectively, deproteinised and
lyophilised. The fragments have been mixed in an equimolar ratio. This marker is
particularly suitable for comparison with PCR fragments (e.g. food diagnostics) and
DNA from restriction samples on agarose gels with sizes between 150 and 2000 base
pairs. The concise distribution of fragments of the marker makes this product ideal
for long and short gel runs with running distances of 70-80 mm (1.5 % agarose
gel). Load 1 µg of the marker per lane of a normal sized agarose gel (approx. 80 ml
Loading buffer is supplied separately.
Number of bands 12
Fragment sizes (bp) 2176; 1766; 1230; 1033; 653; 517; 453; 394; 298; 234; 220; 154
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 1.0 µg per lane
Storage -20°C. Prevent repeated freezing and thawing (> 20x). We recommend to prepare
small aliquots. The lyophilised form is stable even for weeks at ambient temperature!
Package sizes A6927,0050 50 µg

Resuspend the DNA in the sterile-filtered gel loading buffer (supplied with the marker) for 10 minutes at room
temperature by occasional shaking.

DNA
12 Size Marker • © 2010 AppliChem
 DNA Marker Phage Lambda - Sty I A5194

Description Lambda DNA (cI857 Sam 7) Sty I markers are prepared by digesting Lambda
DNA with Sty I, followed by inactivation of the enzyme. The DNA fragments
are then ethanol-precipitated and resuspended in the storage buffer.
Number of bands 11
Fragment sizes (bp) 19329*; 7743; 6223; 4254*; 3472; 2690; 1882; 1489; 925; 421; 74
Storage buffer 10 mM Tris · HCl (pH 8.0), 1 mM EDTA
Concentration 0.2-0.5 µg/µl
Storage -20°C
Package sizes A5194,0005 0.05 mg (= 250 µl)
A5194,0025 0.25 mg (= 1.25 ml)
Please note! The cohesive ends of fragments 1 and 4 (*) may cause the formation of an extra band (23583 bp).
The fragments may be separated by heating to 65°C for 3 minutes before loading the sample onto the gel.

DNA Marker Phage Lambda - BstE II (lyophilised) A4412

DNA size standard made of phage λ DNA for the determination of large DNA fragments (digestion of genomic DNA)
Description Natural lambda DNA was digested with BstE II, deproteinized and lyophilized. This
 arker is suitable for comparisons with fragment sizes between 8400 and 700 base
m
pairs. The best results are achieved after a migration distance of approx. 80-90 mm
on 1.0-1.2 % agarose gels. Each lane of a normal sized gel (approx. 80 ml) should
be loaded with approx. 1 µg of marker. Loading buffer is supplied separately.
Number of bands 14
Fragment sizes (bp) 8454*, 7242, 6369, 5686*, 4822, 4324, 3675, 2323, 1929, 1371, 1264, 702,
224, 117
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 1.0 µg per lane
Storage -20°C
Package size A4412,0100 2 x 50 µg
Please note! The cohesive ends of fragments 1 and 4 (*) may form an additional band (14140 bp). Before loading
onto the gel, the fragments can be separated by heating to 65°C for 5 minutes with subsequent incubation on ice.

marker
© 2010 AppliChem • Size Marker 13
 DNA Marker Phage Lambda - BstE II A5220

Description Phage Lambda (cI857 Sam 7)DNA/BstE II markers are prepared by digesting
L ambda DNA with BstE II, followed by inactivation of the enzyme. The DNA
­fragments are then ethanol-precipitated and resuspended in the storage buffer.
Number of fragments 14
Fragment sizes (bp) 8454*, 7242, 6369, 5686*, 4822, 4324, 3675, 2323, 1929, 1371, 1264, 702,
224, 117
Storage buffer 10 mM Tris · HCl (pH 8.0), 1 mM EDTA
Concentration 0.2-0.5 µg/µl
Storage Store at -20°C
Package sizes A5220,0005 0.05 mg (= 250 µl)
A5220,0025 0.25 mg (= 1.25 ml)
Please note! The cohesive ends of fragments 1 and 4 (*) may cause the formation of an extra band (14140 bp).
The fragments may be separated by heating to 65°C for 3 minutes before loading the sample onto the gel.

DNA Marker Phage Lambda - Hind III (lyophilised) A5589

DNA size standard made of phage λ DNA for the determination of large DNA fragments (digestion of genomic DNA)
Description Natural lambda DNA was digested with Hind III, deproteinized and lyophilized. This
marker is suitable for comparisons with large to medium-sized fragments between
23,000 and 600 base pairs. The best results are achieved after a migration distance of
approx. 80-90 mm on 1.0-1.2% agarose gels. Each lane of a normal sized gel (approx.
80 ml) should be loaded with approx. 0.5-0.8 µg of marker. Loading buffer is
supplied separately.
Number of bands 8
Fragment sizes (bp) 23130*; 9416; 6557; 4361*; 2322; 2027; 564; 125
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.5-0.8 µg per lane
Storage -20°C
Package size A5589,0100 2 x 50 µg
Please note! The cohesive ends of fragments 1 and 4 (*) may form an additional band (27491 bp). Before loading
onto the gel, the fragments can be separated by heating to 65°C for 5 minutes with subsequent incubation on ice.

DNA marker
14 Size Marker • © 2010 AppliChem
DNA Marker Phage Lambda - Hind III A5223

Description L ambda DNA (cI857 Sam 7)/Hind III markers are prepared by digesting Lambda DNA with
Hind III, followed by heat-inactivation of the enzyme. The DNA fragments are then ethanol-
precipitated and resuspended instorage buffer.
Number of bands 8
Fragment sizes (bp) 23130*; 9400; 6557; 4361*; 2322; 2027; 564; 125
Storage buffer 10 mM Tris · HCl (pH 8.0), 1 mM EDTA
Concentration 0.2-0.5 µg/µl
Storage -20°C
Package sizes A5223,0005 0.05 mg (= 250 µl)
A5223,0025 0.25 mg (= 1.25 ml)
Please note! The cohesive ends of fragments 1 and 4 (*) may cause formation of an extra band (27491 bp).
The fragments may be separated by heating to 65°C for 3 minutes before loading the sample onto the gel.

DNA Marker pUC19 - Msp I A5235

Description Digestion of the pUC19 plasmid yields 12 fragments.

Number of bands 12 (9 visible)
Fragment sizes (bp) 501, 489, 404, 331, 242, 190, 147, 111, 110, 67, 34x2, 26
Storage buffer 10 mM Tris · HCl (pH 7.8), 10 mM NaCl, 1 mM EDTA
Concentration 0.2 mg/ml
Storage -20°C
Package sizes A5235,0005 0.05 mg (= 250 µl)
A5235,0025 0.25 mg (= 1.25 ml)
Assay conditions:
1.7 % agarose
DNA Marker pUC19 - Msp I (lyophilised) A3996
DNA size standard for high-percentage agarose gels and polyacrylamide gels
Description Plasmid pUC19 was digested with Msp I, deproteinized and lyophilized. This marker is particu-
larly suitable for comparisons with small PCR fragments and DNA from restriction samples on
polyacrylamide gels or high-percentage agarose gels. The bands at 501/489 bp and 111/110 bp
have approx. double mass and provide rapid orientation after a short migration distance on 2.0-
2.2 % agarose gels. The best results are achieved after a migration distance of approx. 50-70 mm
on agarose gels or 100 mm on 6-8 % polyacrylamide gels. Each lane of a normal sized agarose
gel (approx. 80 ml) should be loaded with approx. 0.5-1.0 µg of marker. Loading buffer is
supplied separately.
Number of bands 12
Fragment sizes (bp) 501, 489, 404, 331, 242, 190, 147, 111, 110, 67, 34, 26
Supplied with 1 ml 10 mM Tris · HCl (pH 7.5); 5 mM sodium acetate; 2 mM EDTA; 10 % glycerol;
loading buffer (1X) 0.02 % bromophenol blue; 0.015 % xylene cyanol
Loading volume 0.5-1.0 µg per lane
Storage -20°C
Package size A3996,0050 50 µg
Please note! Extremely small fragments can only be visualized using high-percentage and high-quality agarose gels (> 2.2
%) with large mass of fragments.

© 2010 AppliChem • Size Marker 15

Prod.-No.
2.1 dyes for nucleic
Description DNA/RNA Dye Complex Excitation/Emission (Detection)
A1398 Acridine orange dsDNA/RNA green fluorescence; 490 nm / 525 nm
ssDNA/RNA red fluorescence
A0691 Crystal violet DNA (purple-red) 590 nm
A1001 DAPI specific binding to AT-Base pairs; 365 nm / 450 nm
intercalation into GC-Base pairs; white-blue fluorescence
A1151 Ethidium bromide* DNA intercalation 260-360 nm or 546 nm / 590 nm
A2388 Malachite green oxalate DNA 626 nm
A1402 Methylene blue DNA, RNA-staining at acidic pH 297 nm / 672 nm
A5595 DNA-Dye Methylene blue DNA 297 nm / 672 nm
A1403 Methyl green specific binding to AT-rich sequences; DNA (green) 638 nm
A0581 Methyl orange (C.I. 13025)
A3918 Nile blue DNA intercalation (blue) 630 nm / 673 nm
A2261 Propidium iodide DNA-intercalator; no uptake into living cells 530 nm / 625 nm
A1406 Pyronin Y DNA/RNA (red) 488-530 nm / 565-574 nm
A3944 Silver nitrate DNA (brown)
A1400 Stains all RNA (blue-violet)
* There are several ready-to-use solutions available!
A1152 Ethidium bromide - Solution 1 % BioChemica
A2273 Ethidium bromide - Solution 0.07 % “dropper-bottle”

* DNA-Dye Methylene blue A5595

Nontoxic solution for the staining of nucleic acids in agarose gels
Amount 10 ml 200-fold methylene blue dye concentrate
Storage 2-8°C
Stability 18-24 months (shake well before use)
Package size A5595,0010 10 ml

Protocol for staining with DNA-Dye Methylene blue:

The DNA marker
Phage Lambda-Hind III
(A5589) was loaded
on the 1st lane (*).
Approx. 50 ml 1X DNA-Dye Methylene blue staining solution are required to stain an agarose
gel with the dimensions of approx. 80 x 60 mm (width x length) and a thick ness of 3-4 mm.
The gel should be trimmed down appropriately, with the edge of a ruler for example, to
remove areas of the gel that were not used for DNA separation. The staining is performed in an
adequately sized dish, in which the gel is floated to a depth of about 5 mm with dye solution.
To prepare the DNA-Dye Methylene blue staining solution for use, 250 µl are dissolved in
50 ml water. The gel is left to stain for about 20 minutes and the dish is shaken occasionally.
To reveal the bands (different staining intensities) the gel is destained with tap water several
times for about 5-10 minutes. The blue-stained DNA bands are clearly visible in transmitted light and can be
measured with a ruler or documented photographically.
Methylene blue does not stain DNA permanently: depending on the thickness of the gel and the storage tempe-
rature, the DNA bands (especially smaller fragments) may lose their staining after some time.

16 Size Marker • © 2010 AppliChem

2.2
acids
Sensitivity recommended working concentration Stock solution / Storage
50 ng 30 µg/ml 1 µg/ml in water; 2-8°C

10 ng 1 µg/ml
70 ng 0.1 µg/ml in water

0.5 ng 0.2-0.5 µg/ml 10 mg/ml in water; 2-8°C

40 ng 0.2 % in 0.4 M NaOAc/0.4 M Acetic acid
40 - 80 ng 200-fold concentrated solution; 2-8°C
1 µg/ml
10 ng 0.0005 % 0.25 % in water
40 ng on agarose; 4 ng on dried gel 1 µg/ml 1 µg/ml in 0.25X TAE buffer (pH 8.0)
1 µg/ml 2-8°C or RT
0.05 % or 1 µg/ml
2.5 ng dsDNA on agarose

Abbreviations
DAPI (4',6-Diamidino-2-phenylindole dihydrochloride); PBS (Phosphate-buffered saline); NaOAc (Sodium acetate)
PBS (Phosphate-buffered saline)

Repeated use of the 1X staining solution

The prepared 1X methylene blue staining solution can be kept in a suitable brown-glass bottle at 2-8°C for
4-6 months and can be used several times. The staining period should be lengthened by about 20 % for the second use, and
by the same again for the third use. After this, the staining capacity of the solution is largely exhausted and a new solution
should be prepared.

Sensitivity
The bands in a 1 kb ladder (loading amount 1 µg) can be stained with no problems. Small fragments (150 bp) can also be
stained. The decisive factors in sensitivity are the thickness of the gel, the gel:staining solution ratio, and the destaining time.
By varying the staining conditions, you can easily determine the best conditions for your system.

Handling
A laboratory coat and gloves should be worn when handling the staining solution.

Precaution
On contact with the skin or clothing, the area should be washed with large amounts of soap and water.

Short protocol
1. Dilute DNA-Dye Methylene blue 200X concentrate down to 1X (250 µl DNA-Dye Methylene blue + 50 ml water)
2. Trim down the agarose gel to the smallest area possible.
3. Incubate the gel (dimensions approx. 80 x 60 mm) with 50 ml DNA-Dye Methylene blue for about 20 minutes;
shake occasionally. The staining solution should cover the gel to a depth of about 5 mm.
Please note: the staining solution can be used several times!
4. Destain repeatedly in water for 5-10 minutes.

© 2010 AppliChem • Size Marker 17

 2.3
Ethidium bromide
is the most commonly used dye for staining DNA during or after polycrylamide or agarose gel electrophoresis.
Excitation with light at a wave lenght of 254 nm is absorbed by the DNA and transferred to ethidium bromide.
Excitation at 366 nm directly excites the dye (3). An aqueous stock solution is prepared at a concentration of
10 mg/ml and employed at 0.2 - 0.5 µg/ml (5). Staining is performed after the gel run (especially PAGE) or
during the gel run (agarose). The latter allows the observation of the gel run by control under UV light. Ethidium
bromide is added to the agarose solution. Please note, that 'prestaining' may lead to artifacts (4).
In another application, ethidium bromide may be used to sensitively quantify DNA by spectrofluorimetry.
dyes for nucleic acids
Excitation at 250 nm with UV light and an emission at 605 nm, the sensitivity is even better as compared to the
dye Hoechst 33258. At a concentration of 0.5 µg/ml EtBr, the detection limit of 10 ng/ml DNA is reached with a
linear range of measurement from 20 to 1250 ng/ml (6).
Caution: Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn when working
with solutions that contain this dye, and a mask should be worn when weighing ethidium bromide.

Ethidium bromide - Solution 1 % BioChemica A1152

Synonym 2,7-Diamino-10-ethyl-9-phenylphenanthridium bromide - Solution
Composition Ethidium bromide: 10 mg/ml
filtered solution
Storage Store the stock solution at 2-8°C protected from light.
Package sizes A1152,0025 25 ml
A1152,0100 100 ml

Ethidium bromide - Solution 0.07 % “dropper-bottle” A2273

Synonym 2,7-Diamino-10-ethyl-9-phenylphenanthridium bromide - Solution
Composition Ethidium bromide: 0.7 mg/ml
filtered solution
Storage Store the stock solution at 2-8°C protected from light.
Description Ethidium bromide acts as a DNA intercalator and has a considerable mutagenic
potential. In order to reduce the possible contact to ethidium bromide AppliChem
provides a ready-to-use solution in a 'dropper bottle'. The concentration of ethidium
bromide solution (0.7 mg /ml) is adjusted so that the addition of one drop (of a
volume of 50 µl) of this solution is sufficient to stain 50 ml agarose gel solution.
Package sizes A2273,0005 5 ml
A2273,0015 15 ml

Literature
(1) Waring, M. (1975) in Antibiotics Vol. III , 141-165 (J.W. Corcoran & F.E. Hahn eds.) Springer-Verlag: Review article about ethidium and propidium.
(2) Lunn, G. & Sansone, E.B. (1987) Anal. Biochem. 162, 453-458 Ethidium bromide: Destruction and decontamination of solutions.
(3) Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press. Cold Spring
Harbor, New York.
(4) Gärtner, U. et al. (1996) Anal. Biochem. 243, 194-196 Apparent degradation of cleaved genomic DNA may be an ethidium bromide prestaining artifact.
(5) Lucey, M.J. et al. (1997) BioTechniques 23, 780-782 Reducing the ethidium bromide quantity in agarose gel electrophoresis.
(6) Bonasera, V. et al. (2007) BioTechniques 43, 173-176 Protocol for high-sensitivity/long linear-range spectrofluorimetric DNA quantification using EtBr.

18 Size Marker • © 2010 AppliChem

protein marker
General Considerations
Size estimation: Prestained protein markers are not recommended for precise determination of the molecular
weight size since their behavior during electrophoresis strongly depends on electrophoresis conditions. For
exact determinations of protein sizes use unlabeled marker proteins, i.e. non-prestained markers.
Protein blotting: The transfer of proteins during blotting depends on their size (e.g. lysozyme is fully transfer-
red after 30 minutes, while myosin requires 2.5 hours @ 1V/cm2).

Prod.
No.
A5238
A5418
A4402
Protein Marker

Protein Marker I (14-116)

Protein Marker II (6.5-200) prestained
Protein Marker III (6.5-200)
Bands

7
8
8
Fragment Sizes [kDa]

116 97.4 66.2 37.6 28.5 18.4 14

200 116 68 43 30 20 14.4 6.5
200 116 68 43 30 20 14.4 6.5
3.1
A3993 Protein Marker IV (10-150) 8 150 100 80 60 40 30 20 10
A8359 Protein Marker V (10-175) prestained 11 175 130 95 70 62 51 42 29 22 14 10.5
A8889 Protein Marker VI (10-245) prestained 12 245 180 135 100 75 63 48 35 25 20 17 11

Protein Marker I (14-116)

3.2 precision A5238
Description The bands of 3 µl marker are easily
visualized with Coomassie staining in a
polyacrylamide gel. Protein Marker I is a
mixture of 7 purified proteins supplied in gel
loading buffer for direct application to
an SDS polyacrylamide gel.
Number of bands 7
Fragment sizes (kDa) 116.0; 97.4; 66.2; 37.6; 28.5; 18.4; 14.0
Loading buffer 50 mM Tris · HCl (pH 6.8),
100 mM dithiothreitol, 2 % SDS,
0.1% bromophenol blue, 10 % glycerol
Assay conditions 3 µl/12 % PAGE
Package size A5235,0500 500 µl

© 2010 AppliChem • Size Marker 19

 Protein Marker III (6.5-200) A4402
Description The bands of the 2.5 µl marker are easily
visualized with Coomassie staining in a
polyacrylamide gel.
Number of bands 8
Protein sizes (kDa) 200.0; 116.0; 68.0; 43.0; 30.0; 20.0;
14.4; 6.5
Assay conditions 5 µl/4-20 % SDS-PAGE gradient gel;
Coomassie staining
Storage -20°C, if stored longer than 1 month.
Please note that repeated freezing and thawing will reduce pro-
duct quality. Preparation of small aliquots is recommended.
Package size A4402,0001 1.0 ml

This is a ready-to-use marker containing acylated proteins.

The product may contain residual iodoacetamide.

precision
Protein Marker IV (10-150) A3993
Application Recombinant protein size marker for gel electrophoresis
Description The bands of 2.5 µl marker are easily
● The proteine markers visualized with Coomassie staining in a
have to be “preheated” polyacrylamide gel.
to room temperature,
to guarantee that all com- Number of bands 8
ponents are dissolved. Protein sizes (kDa) 150; 100; 80; 60; 40; 30; 20; 10
● Apply 1-5 µl of the Assay conditions 10 µl/4-20 % SDS-PAGE gradient gel
marker to a mini-gel
(10 x 10 cm, 1-0.75 mm (Tris-Glycine); Coomassie staining
thick, 7 mm slot). Storage -20°C, if stored longer than 1 month.
Use 1X Laemmli buffer Please note that repeated freezing and
to dilute this marker, thawing will reduce product quality.
if necessary.
● To minimize myosin Preparation of small aliquots is
aggregation, the aliquot to recommended. Thawed aliquots are stable
be loaded onto the gel for one week at +4°C.
may be heated to 95°C Package size A3993,0500 500 µl
for 1-2 minutes.

This is a ready-to-use marker containing recombinant proteins.

The product may contain residual iodoacetamide.

20 Size Marker • © 2010 AppliChem

 prestained
Protein Marker II (6.5-200) prestained A5418
Prestained protein size marker for gel electrophoresis
Description This is a ready-to-use marker containing
covalent prestained proteins. The product
contains formamide.
Number of bands 8

3.3
Protein sizes (kDa) 200.0; 116.0; 68.0; 43.0; 30.0; 20.0;
14.4; 6.5
Assay conditions 10 µl/4-20 % PAGE gradient gel
Tris-Glycine; left lane prestained but not
Coomassie-stained; right lane prestained
and additional Coomassie staining.
Storage -20°C, if stored longer than 1 month.
Please note that repeated freezing and
thawing will reduce product quality. Pre -
paration of small aliquots is recommended.
Package size A5418,0250 250 µl

Protein Marker V (10-175) prestained A8359

Prestained protein marker for gel electrophoresis and Western
blotting. Magenta Marker
Description Ready-to-use prestained protein size marker. Supplied in loading buffer.
Number of bands 11
Protein sizes (kDa) 175; 130; 95; 70; 62; 51; 42; 29; 22; 14; 10.5
Three reference proteins coupled with a blue dye for easy identification:
approx. 10, 40, and 90 kDa.
Storage At 2-8°C for max. 3 months; at -20°C for long term storage.
Package size A8539,0250 250 µl

© 2010 AppliChem • Size Marker 21

 Protein Marker VI (10-245) prestained A8889
Prestained protein marker for gel electrophoresis and Western blotting. Blue-Green-Red Protein Marker
Description Protein Marker VI prestained is a three-color protein standard with 12 prestained
proteins covering a wide molecular weight range from approx. 10 to 245 kDa.
Proteins are covalently coupled with either a blue chromophore or one green
(25 kDa) and one red dye (75 kDa), respectively. Gel run may be monitored
during protein separation on SDS-PAGE (Tris-glycine buffer, “Laemmli system”).
The main applications are the monitoring of protein migration during
SDS-polyacrylamide gel electrophoresis, the verification of Western transfer
efficiency on membranes (PVDF, nylon, or nitrocellulose), and the sizing of
proteins on SDS-polyacrylamide gels and Western blots. The size marker is
supplied ready-to-use in gel loading buffer.
Number of bands 12
Protein sizes (kDa) 245; 180; 135; 100; 75; 63; 48; 35; 25; 20; 17; 11
Package size A8889,0500 500 µl

Recommendations for Loading

1. Thaw the ladder either at room temperature or at 37-40°C for a few minutes to dissolve pre-
cipitated solids. Do not boil!
2. Mix thoroughly to ensure the solution is homogeneous.
3. Load the following volumes of the ladder on SDS-polyacrylamide gel: 5 µl per well for mini-gels,
2.5 µl per well for blots; 10 µl per well for large gels, 5 µl per well for blots.

3.3 prestained

22 Size Marker • © 2010 AppliChem

 Sometimes,
less is more!

For The Sake Of For Better Results

The Environment Changing the composition has no negative
influence on the performance of the products.
Many reagents prepared in biomedical research
labs are ideal nutrient broths for unwanted
For Your Safety With CrossDown and all other immunoassay
buffers you are even in a better mood and your
germs (bacteria, fungi). To prevent their growth, immunoassays show a better quality.
reagents are either autoclaved, sterile filtered, or We would like to keep you healthy so that you
antibiotics / antimycotics or toxic substances are stay our customer. FYI: Thimerosal is classified
added. One of these additives is Thimerosal, a as toxic. The lethal dose (rat, s.c.) is 9 mg/kg,
mercury-containing molecule which is dangerous compared to ethidium bromide with a lethal dose
for the environment. Our original immunoasssay (mouse, s.c.) of 110 mg/kg. In some countries,
buffer contained this chemical too, but now we Thimerosal is a forbidden additive.
have replaced it by the nontoxic ProClin® 300.
For the sake of the environment.

© 2010 AppliChem • Size Marker 23

4.1
dyes for protein gelsProd.-No. Description
A2176 Bismarck brown R
Comment
increases sensitivity of Coomassie®
Brilliant Blue R-250 staining
Absorption maxima
lmax. 468 nm

A3480 Coomassie® Brilliant Blue G-250 stains ampholytes in IEF gels too!*

A1092 Coomassie® Brilliant Blue R-250 stains ampholytes in IEF gels too!* lmax. with protein 549 nm,
w/o protein 555 nm
A0822 Eosin Y reversible staining

A1346 Eriochrome black T 560 nm

A1401 Fast Green FCF dye complex in acidic pH range lmax. (pH 8.3) 615; (MeOH) 620 nm
lmax. (with protein) 635 nm
A2385 Kongo red staining in the acidic pH range
A2388 Malachite green oxalate Phosphatase staining in gels

Nile red
A1405 Ponceau S acidic diazo dye lmax. (H2O) 517 - 823 nm

A7808 Proteo-Dye RuBPS lmax. 617 nm

A3930 Rhodamine B $ 560 nm

A3972 Silver nitrate brown

A1400 Stains all ** stains different types of proteins lmax. for BSA 515 nm
in different colors
Zinc-Imidazole reversible staining

Abbreviations: BSA (Bovine Serum Albumin); CBB (Coomassie® Brilliant blue); MeOH (Methanol);
RuBPS (Ruthenium(II)tris(bathophenanthroline disulfonate)); TCA (Trichloroacetic acid); & only in combination with Coomassie®!
$ detection of phosphoproteins; formation of insoluble phosphate complexes (sensitivity: 0.1 nM)

Coomassie® Brilliant blue R-250 (C.I. 42660) A1092

Synonym Brilliant blue R, Xylenebrilliantcyanine
Order No. Quantity Formula M CAS-No.
A1092,0010 10 g C45H44N3NaO7S2 825.98 g/mol 6104-59-2

4.2
A1092,0025 25 g
A1092,0100 100 g
® registered trademark of Imperial Industries PLC

Coomassie® Brilliant Blue R-250 is one of the most commonly used stains for proteins, after their separation by polyacrylamide gel electro-
phoresis. The protein-dye complex has an absorption maximum at 549 nm, the dye without protein at 555 nm (in 0.01 M citrate buffer,
pH 3). The intensity in staining of proteins probably depends on the basicity of a protein. Per positively charged amino acid approximately
1.5-3 molecules of Coomassie® will be bound. This variation complicate the exact protein determination with albumin as a standard, since this
protein contains more basic amino acids than many other proteins .
There do exist many protocols for sensitive staining procedures with Coomassie® (e. g. ref. 3, 4). The sensitivity reaches a limit at 25 ng protein.
We recommend the following protocol:
I. Staining solution: 0.1 % Coomassie® Brilliant Blue R-250 (Prod.-No. A1092), 20 % methanol (or ethanol), 10 % acetic acid.
The SDS gel (without 'stacking gel') is stained for 1 hour at 60°C or for 2 hours at 50°C or over night at RT.
II. Destaining solution: 20 % methanol (or ethanol) and 10 % acetic acid. Destain the gel for 3-4 hours at 50 - 60°C. Add some sponges.
Subsequently wash the gel for 15 minuts in water and dry under vacuum at 60°C for 2-3 hours.

24 Size Marker • © 2010 AppliChem

Sensitivity Recommended Concentration
25 ng & Coomassie Brilliant blue R-250 (0.2 % w/v)
Bismarck brown R (0.05 % w/v)
< 0.5 ng Mix 80 ml of the stock solution
with 20 ml of MeOH just before use.
10 ng 0.1 % in 20 % MeOH, 10 % Acetic acid
Stock solution
Solvent MeOH : Acetic acid : Water (40:7:53);
dissolve dyes separately, then mix 1:0.75 (v/v)
0.1 % w/v CBB in 2 % w/v Phosphoric aicd,
10 % w/v Ammonium sulfate (Do not filter!)

similar to CBB R-250 0.25 % (w/v) in 0.1 N NaOH prepare staining solution fresh daily;
may be used for several gels;
10 ng 0.01 % 0.02 % in 40 % MeOH / 7 % Acetic acid;
in combination with Rhodamine B
400 ng 0.25 % (w/v) in 10 % Acetic acid up to 1 %
(w/v) in 7 % Acetic acid
less sensitive than CBB 0.1 % (w/v) in 0.2 M Acetate buffer (pH 3.5) 1 % (w/v) in distilled water
Staining solution = Solution 1: 0.15 g Malachite green oxalate in 300 ml Water;
3 Vol. Solution 1 + 1 Vol. Solution 2 Solution 2: 4.2 g Ammonium molybdate tetrahydrate in 100 ml
(stable for 3 weeks; filter prior to each use) 5 N HCl;
5 ng
500 ng 0.1 - 0.5 % in 3 % TCA or 1 ml Acetic acid prepare fresh prior to use
glacial ad 100 ml Water
2-5 ng 1 µM 1 mM
10 ng 0.01 % 0.02 % in 40 % MeOH / 7 % Acetic acid; in combination with
Eriochrome black T
2-5 ng
like CBB 0.005 % 0.1 % in Formamide or 5.6 mg/10 ml in 50 % Dioxane;
prepare fresh
10-20 ng SDS-PAGE
40-80 ng native PAGE

* Allen, R.E. et al. (1980) Anal. Biochem. 104, 494-498. Staining Proteins in Isolelectric Focusing Gels with Fast Green.
** stains e.g. sialoglycoproteins and phosphoproteins blue, almost all other proteins red.

Coomassie® Brilliant blue G-250 (C.I. 42655) A3480

Synonym Brilliant blue G
Order No. Quantity Solubility (20°C) Formula M CAS-No.
A3480,0010 10 g ~10 g/L (H2O) C47H48N3NaO7S2 854.04 g/mol 6104-58-1
A3480,0025 25 g
A3480,0100 100 g
® registered trademark of Imperial Industries PLC

The assays of Neuhoff et al. (Neuhoff, V. et al. (1988) Electrophoresis 9, 255-262) have shown that stainng with
Coomassie® G-250 is more sensitive than with R-250. For more informations see A1092.

© 2010 AppliChem • Size Marker 25

 Proteo-Dye Blue-Vis A6810

Synomym Protein dye in the visible range

Storage 2-8°C protected from light
HS-No. 38220000
Package size A6810,1000 1L
1 L is sufficient for approx. 30 minigels, since 3x reusable
detection limit 3-5 ng/mm²
reversible staining of proteins

Protocol for staining of gels with Proteo-Dye Blue-Vis:

SDS-PAGE minigels (1 mm thickness, 10 % acrylamide, Tris-glycine buffer system) are
treated for 1 hour with 30 % v/v methanol after electrophoresis to remove the excess of
SDS and to fix the proteins in the gel matrix. The gels are stained for 2 hours in a volume
of 100 ml Proteo-Dye Blue-Vis. After this, the gels are washed with acetate buffer (0.2 M;
pH 4.5), containing 20 % v/v methanol for 90 - 120 minutes until the dark red background
is reduced to a weak pink background in contrast to the blue protein bands. The gels are
documented with the help of a video system (transilluminator 312 nm, white light plate) or
on a scanner in the transmission mode.

4.3 dyes
Proteo-Dye Red-Fluo A6803

Synomym Protein dye with red fluorescence

Storage 2-8°C protected from light
HS-No. 38220000
Specification Emission maximum: 630 nm
Exicitation wave lenght: 312 nm
Package size A6803,1000 1L
1 L is sufficient for approx. 30 minigels, since 3x reusable
detection limit 1-3 ng/mm²
reversible staining of proteins
more sensitive than most other products available in the market

Protocol for staining of gels with Proteo-Dye Red-Fluo:

SDS-PAGE minigels (5x8 cm, 1 mm thickness, 10 % acrylamide, Tris-glycine buffer system)
are fixed for 30 minutes in a solution of 7.5 % v/v acetic acid, 20 % v/v ethanol. After this,
the gels are stained with Proteo-Dye Red-Fluo (100 ml) for 2 - 3 hours. The background
fluorescence can be removed by repeated washing with the fixation solution (7.5 % v/v
acetic acid, 20 % v/v ethanol; 3 - 4 washing steps, 30 minutes each). The documentation
of the gels is carried out with a video system (transilluminator 312 nm, adapting filter
590 nm).

26 Size Marker • © 2010 AppliChem

Proteo-Dye Green-Fluo A6794

Synomym Protein dye with green fluorescence

Storage 2-8°C protected from light
HS-No. 38220000
Specification Emission maximum: 520 nm
Excitation wave lenght: 365 nm
Package size A6794,1000 1L
1 L is sufficient for approx. 30 minigels, since 3x reusable
detection limit 3-5 ng/mm²
reversible staining of proteins
suitable for gels and for blots

Proteo-Dye Green-Fluo is a fluorescence protein dye, based on a metal-chelate complex in

conjunction with a detergent in submicellar concentration in an aqueous solution. This dye
solution enables to detect very small amounts of proteins and the staining is fully reversible.
Use the dye solution up to three times!

Protocol for staining of gels with Proteo-Dye Green-Fluo:

SDS-PAGE minigels (5x8 cm; 1 mm thickness, 10% acrylamide, Tris-glycine buffer system)
are postelectrophoretically treated for 30 minutes with a methanol solution (30 %, v/v)
while gently shaking (100 rpm). After this, the gels are stained for 2 hours in a volume of
100 ml Proteo-Dye Green-Fluo. Reduction of the background fluorescence is achieved by
repeated washing steps (3-4 times, 30 min. each) with 30 % v/v methanol. The gels are
documented with a video system (transilluminator 365 nm, adapting filter 520 nm).

Protocol for staining of blots with Proteo-Dye Green-Fluo:

By staining with Coomassie or silver the proteins in the gel become irreversibly de­naturated
and are difficult to transfer to blotting membranes. Metal chelate staining methods are
reversible, resulting in good preconditions for the following blot transfer. Blots were
performed by a typical semi-dry method. After the blotting process, the blots were washed
in distilled water for 1 hour and dried at room temperature. Blots were then stained with
Proteo-Dye Green-Fluo for 2 hours, briefly washed in 30 % v/v ethanol and dried again.
Only in the dried state, intensely gree n fluorescent protein bands become visible in UV
toplight (365 nm UV light) and are documented with a video system (adapting filter 420
nm). The stain may be easily and fastly removed just by rinsing in 30% v/v ethanol.

for protein gels

© 2010 AppliChem • Size Marker 27


2-D gels of E. coli proteins stained with (1) SYPRO
Ruby© and (2) with RuBPS. For details see Reference 2.
Literature
[1] A comparison between Sypro Ruby© and
ruthenium(II) tris(bathophenanthroline disulfonate)
as fluorescent stains for protein detection in gels.
(Rabilloud, T. et al. (2001) Proteomics 1, 699-704)
[2] Improved ruthenium(II)
tris(bathophenanthroline disulfonate) ­staining
and destaining protocol for a better signal-to-
background ratio and improved resolution.
(Lamanda, A. et al. (2004) Proteomics 4, 599-608)
28 Size Marker • © 2010 AppliChem
Proteo-Dye RuBPS A7808
Synomym Ruthenium(II) tris(bathophenanthroline disulfonate) tetrasodium salt
Formula C72H42N6Na4O18RuS6
Molecular weight 1664.58 g/mol
CAS-No. [301206-84-8]
Concentration (in H2O) 1 mM
lmax. Emission (in H2O) 617 nm
lmax. Exitation 277, 437, 460 nm
Storage 2-8°C, protected from light
recommended working solution 1 : 1000 dilution (1 µM final concenrtation)
Stability 2 years
Package size A7808,0001 1 ml
Comment
RuBPS is a fluorescence dye for protein detection in e.g. SDS- and 2-D-gels. It provides the high
sensitivity of silver staining without its drawbacks. The excellent contrast, good linearity and
homogeneity made this dye the staining reagent of choice in proteomics. A minimal interference
with MALDI-TOF analysis is observed and compatibility with MS/MS analysis is given. The photo-
chemically stable dye is excited with UV-light of the wavelength 473 or 488 nm blue laser. A 532
nm laser may be used as well, albeit with reduced ­efficiency. It is of advantage that the excitation
wavelength of RuBPS is longer than tose of the aromatic amino acids.
The information given in terms of formula, molecular weight and CAS number refer to the
raw material! The concentrate supplied is diluted 1 : 1000 (1 ml ad 1 L) resulting in an
1 µM working solution.
Special features of Proteo-Dye RuBPS
• Provides the sensitivity of silver-staining without its drawbacks
(limited dynamic range, inhomogeneity and interference with
MS analysis)
• Carefully purified product offers much improved signal-to-
background ratio
• Excellent contrast in gel images enables protein spots to be
detected more easily by image processing software
• High sensitivity
• Good linearity and homogeneity
• Minimal interference with MALDI-TOF analysis
• Compatible with MS/MS analysis, provided that cleaning of the
sample with reverse phase adsorption is carried out
Protocol for gel staining / destaining
(according to Ref. 2)
1. Prepare a 1 µM Proteo-Dye RuBPS solution by diluting the concentrate 1000-fold
(e.g., 1 ml to 1 L)
2. Fix the gel in 30 % ethanol, 10 % v/v acetic acid overnight.
3. Rinse the gel in 20 % ethanol v/v for 30 min and repeat 3 times.
4. Incubate the gel in 1 µM Proteo-Dye RuBPS solution for 6 h.
5. Equilibrate the gel in water for 10 min and repeat once (a first scan is possible at this
stage).
6. Destain the gel with 40 % ethanol / 10 % acetic acid v/v for 15 h.
N.B. for low protein concentration the destaining time might be too long. In such cases, shor-
ter destaining times may optimize the procedure resulting in greater sensitivity.
7. Equilibrate the gel in water for 10 min, repeat once and scan.
 related produc t s
Electrophoresis Reagents Prod. No.
Acrylamide Molecular biology grade A3812
Bisacrylamide Molecular biology grade A3636
Agarose Basic A8963
Agarose low EEO (Agarose Standard) A2114
Agarose MP A1091
Loading buffer DNA I A3144
Loading buffer DNA II A2571
Loading buffer DNA IV (for Agarose gels) A3481
TAE buffer (50X) A1691
TAE buffer (10X) A1416
TBE buffer (10X) A0972

Transfer membranes Prod. No.

Reprobe Nitrocellulose supported 0.22 μm Transfer membrane A5237
Reprobe Nitrocellulose supported 0.45 μm Transfer membrane A5242
Pure Nitrocellulose unsupported 0.22 μm Transfer membrane A5250
Pure Nitrocellulose unsupported 0.45 μm Transfer membrane A5239
Pure Nylon Neutral Transfer membrane 0.22 μm (30 cm x 3 m) A4399
Pure Nylon Neutral Transfer membrane 0.45 μm A5248
Reprobe Nylon Positively charged Transfer membrane 0.45 μm (30 cm x 3 m) A5255
PVDF-Star Transfer membrane 0.45 μm A5243

Other Related Products Prod. No.

Chemiluminescence Detection Kits for Horseradish Peroxidase
Cheluminate-HRP PicoDetect A3417
Cheluminate-HRP FemtoDetect A7807
Cheluminate-HRP FemtoDetect Plus A7879
Boric acid Molecular biology grade A2940
EDTA disodium salt dihydrate Molecular biology grade A2937
Tris ultrapure A1086

Transfer Membranes
AppliChem supplies a range of transfer membranes designed
and tested speci­fically for RNA, DNA and protein analysis.
AppliChem also provides our customers with tried
and proven protocols developed to obtain consistent repro­ducible
and dependable results when used with AppliChem membranes.
22 protocols and all types of membranes – all from one source.

© 2010 AppliChem • Size Marker

A26, E 4t Matthes + Traut · Darmstadt

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