S K Patel College Of Pharmaceutical Education & Research

Ganpat University

(
Northern blot for RNA

Western blot
for Proteins
Proteins.

BY,
Nirmal Patel
Southern blot for DNA
DNA.
M Pharm (Bio Tech)
 The Western blot is an analytical
technique used to detect specific
proteins in a given sample of
tissue homogenate or extract.
-može da se koristi za dokazivanje At ili Ag

-osetljiva i specifična tehnika (ELISA - specifičnija)

The name Western blot was given to the technique by W. Neal Burnette.
 WB
It uses gel electrophoresis to separate
native or denatured proteins
by the 3-D structure of the protein by the length of the polypeptide
(native conditions) (denaturing conditions)
Steps in a Western blot:
Contents:
- Tissue preparation

- Gel electrophoresis

- Transfer

- Blocking

- Detection - Two step detection

- One step detection

- Analysis - Colorimetric detection

- Chemiluminescent detection
- Radioactive detection
- Fluorescent detection
 Tissue preparation:
- Samples may be taken from
- whole tissue,
tissue from cell culture
- bacteria
bacteria, virus or
- environmental samples.
samples
Cells may also be broken open
by one of the mechanical methods.
- first broken down mechanically
blender (for larger sample volumes),
homogenizer (smaller volumes), or
by sonication
sonication..
detergents, salts, and buffers
- Biochemica
Biochemicall techniques { Anti Protease and phosphatase }
- mechanical techniques - various types of filtration and centrifugation
centrifugation.
- to encourage lysis of cells and to solubilize proteins,
may be employed :
detergents, salts, and buffers
SDS - Sodium Dodecyl Sulfate (aa detergent used in many western blotting solutions)

- to prevent the digestion of the sample by its own enzymes :

Anti Protease and phosphatase

- When need to avoid protein denaturing

tissue preparation is often done at cold temperatures
 Tissue Preparation

Rastvori za denaturaciju sadrže -HCl

-SDS
-Merkaptoetanol
-Bromfenol plavo → Koriste se za vizuelizaciju kretanja
proteina kroz gel tokom izvođenja
-Pyronin → elektroforeze.
-Ureu
-Glicerol
 Gel electrophoresis:
- The proteins are separated using gel electrophoresis.
- Separation of proteins may be done by
isoelectric point (pI
(pI)) - - - IEF razdvajanje (pH 3.5-10)
molecular weight * - - - SDS razdvajanje
electric charge,
charge or
a combination of these factors.

- most common type of gel electrophoresis

SDS--PAGE (SDS polyacrylamide gel electrophoresis)
SDS

- maintains polypeptides in a denatured state

state, thus allows
separation of proteins by their *molecular
molecular weight tj po broju AK.
AK
 Priprema gela
- Vinil polimerizacija monomera akrilamida (AA) u
duge lance poliakrilamida koji su povezani manjim
količinama komonomera NN’-metilen-bis-
akrilamida (BIS A) (NH ) S O 4 2 2 8

- Katalizator polimerizacije je – amonijum persulfat

(za dejstvo je potrebna bazna sredina pa se dodaje baza TEMED)
TEMED
- Rezultat – gel mrežaste strukture
- Veličina pora se reguliše koncentracijama AA i BIS A
 Priprema gela
• Parametri

T% • Masa AA i BIS A u 100ml rastvora gela [gr/ml]

C% • Odnos mase BIS A u odnosu na ukupnu masu AA i BIS A

• Voditi računa o puferu

puferu:
– pH Utiču na brzinu i rezoluciju
– Molarnosti razdvajanja
• Sobna temperatura proteina
*The concentration of acrylamide determines the resolution of the gel.
- the greater the acrylamide concentration
the better the resolution of lower molecular weight proteins.
proteins
-The lower the acrylamide concentration
the better the resolution of higher molecular weight proteins.
proteins

* Samples are loaded into wells in the gel.

* One lane is usually reserved for a marker or ladder
ladder, (mixture of proteins having
defined molecular weights,
weights, typically stained so as to form visible, coloured bands).

* When voltage is applied along the gel,

proteins migrate into it at different speeds. “Apply 15 µL of the
(different electrophoretic mobilities
mobilities).
). molecular weight standards
to one or two wells...”
Polymerized gel:
Polyacrylamide Gel
aka “preparativni gel” – se ne boji jer boja
-Resolving gels made in može uticati na elekroforetska svojstva
6%, 10%, 12%, 18%. ispitivanih proteina.
-Stacking Gel up to 5%
was added to gel and then the wells are created.

The percentage chosen

depends on the size of the protein
that one wishes to identify (detect or
probe) in the sample.
The smaller it is the bigger the percentage

Najčešće se koristi VERTIKALNA

elektroforeza

Postoji: 1. gel za koncentrovanje

2. gel za separaciju
Bazenčić se puni sa tzv running buffer (SDS, Glycine, baza), a zatim se dodaje
ispitivani protein rastvoren u denaturing buffer-u

1. Gel za koncentrovanje
-blago kiseo (pH 6.5)
-niska koncentracija AA (5%) → porozan

-proteini se slabo razdvajaju ali su trake (linije)

jasno definisane

2. Gel za separaciju
- bazan (pH 8.8)
- viša koncent. AA (6-18%) → kanali (pore) su uži

-Proteini koncentrovani u gornjem gelu, prolaze

kroz uske pore separatnog gela koji omogućava
lakše i brže kretanje manjim proteinima.

Bojenje gela se izvodi nakon izvođenja WB

da bi bili sigurni da je protein prešao na membranu!
(razlikovati bojenje membrane!!!)
WB se izvodi u 3 faze:
1. Ag se elektroforezom u poliakrilamidnom gelu rastavi na
sastavne delove koji se u gelu vide kao zasebne linije.

2. Gel se prekriva nitroceluloznom (ili dr.) membranom i

stavlja između dva filtera natopljena rastvorom pufera.
Proteini se prenose sa gela na membranu.

3. Membrana se uranja u rastvor At za pojedine Ag koja su

označena nekim enzimom. Nakon uranjanja i inkubacije,
trake se ispiraju da bi se uklonila nevezana At. Trake se
zatim uranjaju u rastvor supstrata. Supstrat se razgrađuje
i boji linije u kome su konjugovana At reagovala sa Ag.
 Loading the SDS-
SDS-PAGE gel
Sodium Dodecyl Sulfate Polyacrylamide Gel electrophoresis.
-SDS linearizes all proteins and gives net negative charge
dolazi do razdvajanja proteina po molekulskoj težini

SDS - Sodium Dodecyl Sulfate

(a detergent used in many
western blotting solutions)

(pore)

Veličina pora zavisi od koncentracije

akrilamida (AA) i bis-akrilamida (BIS A)
 -The Pink Strands are the Denatured Proteins.
Proteins See varying sizes

-They Are traveling to the + since they have – charge due to SDS.
----------------
----------------
-These strands go throught the tunnel and are seperated by
their
size!!!
size !!! tj.
molekulske težine

Denatured
Proteins
Proteins here are actually linearized.

Boja:
-Bromphenol blue
-Pyronin

•Boja ne utiče na uzorak,

•putuje
putuje zajedno sa njim
•omogućava praćenje
toka elektroforeze
 Transfer
- In order to make the proteins accessible to antibody detection:
they are moved from within the gel onto a membrane made of
nitrocellulose ,
polyv (PVDF)) or
olyvinileden difluoride (PVDF
nylon.
The membrane is placed on top of the gel,
gel and a stack of filter
papers placed on top of that.
that

- The entire stack is placed in a buffer solution which moves up the paper by
capillary action, bringing the proteins with it. it

- Another method for transferring the proteins is called electrobloting and

uses an electric current to pull proteins from the gel into membrane.
All varieties of membrane are chosen for their
non--specific protein binding properties
non
(i.e. binds all proteins equally well).

Protein binding is based upon hydrophobic interactions,

interactions,
as well as charged interactions between the
membrane and protein.

- Nitrocellulose membranes are cheaper than

PVDF, but are far more fragile and
do not stand up well to repeated probings
probings.
One major difference between
nitrocellulose and PVDF membranes relates to the
ability of each to support "stripping" antibodies off and
reusing the membrane for subsequent antibody probes.

PVDF is sturdier, and can be more reused.

Another difference is that, unlike nitrocellulose, PVDF

ethanol isopropanol or
must be soaked in 95% ethanol,
methanol before use.
PVDF membranes also tend to be thicker and more
resistant to damage during use.
use
The uniformity and overall effectiveness of transfer
of protein from the gel to the membrane can be
checked by staining the membrane with ponponcceau S or
staining the gel with coomassie blue or SILvEr.

Ponceau S - češća jer je osetljivija, i rastvorljiva u

vodi pa je kasnije lakše obezbojiti membranu
(reverzibilno bojenje nitroceluloze).
Coomassie blue se koristi samo za bojenje gela tj za bojenje proteina u
gelu (snažno se vezuje za proteine, a ne za AA). Bojenje membrane
nije reverzibilno. Koristi za sagledavanje efikasnosti transfera
proteina ili za sagledavanje separacije u gelu ako ne postoji namera
za transfer na membranu.
Six general protein stains: Ponceau S colloidal gold
Coomassie blue colloidal silver
amido black India ink
Blotting used to
transfer the Analyzed
samples from the through
gel on to a probing
membrane such with
as a nylon nucleic acid
membrane or probes or
nitrocellulose antibody
membrane. probes..
probes
 *Blokada preostalih slobodnih mesta na membrani sa inertnim proteinima.

steps must be taken to prevent interactions between the

membrane and the antibody used for detection of the target
protein (since the antibody is a protein itself).
itself)

Blocking of non-specific binding is achieved

by placing the membrane in a dilute solution of protein

typically Bovine Serum Albumin (BSA) or non

ovine non--fat dry milk
(both are inexpensive),
inexpensive)
with a minute percentage of detergent such as Tween20
Tween20.
* The protein in the * This reduces
dilute solution "noise" in the
attaches to the final product of
membrane in all the Western blot,
places where the leading to clearer
target proteins have results, and
results
not attached. eliminates false
positives.
positives
 - Two step
- One step Detection
the membrane is "probed" for the protein of interest with a modified antibody
enzyme which when exposed to an
which is linked to a reporter enzyme,
appropriate substrate drives a colourimetric reaction and produces a colour
colour.

- Two step:
step: 1. 2.
Detection -Biotin ili
+ supstrat
-Enzim (AP, HRP)

Primary antibody {Antibodies are generated when a host species or immune

cell culture is exposed to the protein of interest (or a part thereof) }.

A dilute solution of primary antibody (generally between 0.5 and 5 micrograms/mL

micrograms/mL)) is
incubated with the membrane under gentle agitation for anywhere from 30 minutes
to overnight at different temperatures.
temperatures

The solution is composed of buffered saline solution with a small percentage of

detergent, and sometimes with powdered milk or BSA.
detergent
Secondary antibody {After rinsing the membrane to remove
unbound primary antibody, the membrane is exposed to
another antibody, directed at a species-specific portion of the
primary antibody }.

The secondary antibody is usually linked to biotin or to a reporter

enzyms such as alkalin phosphatase or horseradish peroxidase
peroxidase.
This means that several secondary antibodies will bind to one
primary antibody and enhance the signal.

Most commonly, a horseradish peroxidase-linked secondary is

used to cleave a chemiluminescent agent
agent, and the reaction
product produces luminescence in proportion to the amount of
protein.
protein
A cheaper but less sensitive approach utilizes a
4-chloronaphthol stain with 1% horseradish peroxidase
peroxidase;
reaction of peroxide radicals with 4-chloronaphthol produces
a dark brown stain that can be photographed
without using specialized photographic film.

- One step
Historically, the probing process was performed in two steps
because of the relative ease of producing primary and
secondary antibodies in separate processes.
one-step probing systems that would allow the process
one-
to occur faster and with less consumables.
consumables

This requires a probe antibody which both recognizes the

protein of interest and contains a detectable label, probes
which are often available for known proteins tags.
 Analysis :
In practical terms, not all Westerns reveal protein only at
one band in a membrane.

Size approximations are taken by comparing the stained

bands to that of the marker or ladder loaded during
electrophoresis.
electrophoresis

The process is repeated for a structural protein,

protein such as
actin or tubulin, that should not change between
samples.

This practice ensures correction for the amount of total

protein on the membrane in case of errors or
incomplete transfers.
Colorimetric detection :
This method depends on BCIP/NBT Alkaline Phosphatase Substrate Solution
incubation of the Western blot with a substrate
that reacts with the reporter enzyme (such as peroxidase or AP)AP)
that is bound to the secondary antibody.

This converts the soluble dye

into an insoluble form of a
different color that PRECIPITATES next to the enzyme
and thereby stains the membrane.

Protein levels are evaluated through

densitometry or spectrophotometry.
 Chemiluminescent substrates for
Chemiluminescent detection : horseradish peroxidase (HRP)
are two-component systems
consisting of
1. a stable peroxide solution and
2. an enhanced luminol solution.
This methods depend on
incubation of the Western blot with a substrate
that will luminesce when exposed to the reporter on the 2° Ab
Ab..

The light is then detected by photographic film or CCD cameras

cameras.

The image is analysed by densitometry.

Newer software allows further data analysis such as molecular

weight analysis if appropriate standards are used.
Radioactive detection :

Radioactive labels do not require enzyme substrates,

substrates
but rather allow the placement of medical X-X-ray film
directly against the western blot which develops as it
is exposed to the label and creates dark regions
which correspond to the protein bands of interest

very expensive, health and safety risks are high

Fluorescent detection :

The fluorescently labeled probe is excited by light

and the emission of the excitation is then
detected by a photosensor such as CCD camera.
camera

Allows further data analysis such as molecular

weight analysis and a quantitative western blot
analysis.
analysis

the most sensitive detection methods for blotting

analysis.
 Medical diagnostic applications:
• Detekcija At
Inkubacija traka sa ispitivanim serumom, a
potom sa anti-At koja su konjugovana sa enzimom.
Na kraju se dodaje supstrat

• Detekcija Ag
U smeši proteina dobijenih liziranjem μ.o. (k.ć. sa
virusom, bakterije?), identifikuju se Ag upotrebom At
obeleženih enzimima.
Npr. Dg HIV infekcije analizom seruma na At:
 Medical diagnostic applications:
- definitive test for
Bovine Spongiform Encephalopathy (BSE).

-Some forms of Lyme disease testing employ Western blotting.

(The confirmatory HIV test)

test)

-Western blot can also be used as a confirmatory test for Hepatitis B infection.
Advantages
Specific interaction of antibody and antigen can
be directly visualized.

Disadvantages
Technically demanding
Expensive
Subject to interpretation
Presence or absence of bands
Intensity of those bands
 When should WB be used?
WB assay should not be used as a - screening test.

WB should be viewed as a supplemental test which can be

used to confirm positive results obtained from EIA
EIA**
*Enzyme immunoassay

HOWEVER:

Specificity is less than that of EIA !!!

A significant number of indeterminate blots are seen in low risk
populations