Western blotting is a technique used to detect specific proteins in a complex protein mixture. It involves transferring proteins separated by gel electrophoresis onto a membrane and using antibodies to identify a target protein. The key steps are sample preparation, gel electrophoresis to separate proteins, protein transfer to a membrane, blocking to prevent nonspecific antibody binding, primary and secondary antibody probing, and detection of the target protein with an enzyme substrate reaction. Western blotting is widely used to detect viral proteins, characterize antibodies, and study the immune response.

1. WESTERN BLOTTING
PRESENTED BY: RAVI KUMAR
M PHARMACY(1st SEM)
DEPARTMENT OF PHARMACOLOGY
G H G KHALSA COLLEGE OF PHARMACY

2. BLOTTING
Blots are techniques for transferring DNA , RNA and
protein onto a carrier so they can be separated and
often follows the use of a gel electrophoresis
The These powerful techniques allow us to identify
and characterize specific molecules in a complex
mixture of related molecules.
Some of the more common techniques include:
 Southern blotting (DNA)
 Northern blotting (RNA)
 and Western blotting (for protein)

4. WESTERN BLOTTING
 Western blotting, also known as immunoblotting or
protein blotting, is a technique used to detect the presence
of a specific protein in a complex protein mixture.
 It is a core technique in cell biology, molecular biology,
virology and others.
 Western blots have become one of the most common
analytical tools for the
 Detection of viral proteins.
 Characterization of monoclonal and Polyclonal antibody
preparations.
 To determining the specificity of the immune response to
viral antigens

7. WESTERN BLOTTING PROCEDURE
FLOW CHART

8. SAMPLE PREPERATION
 All sources of protein, from single cells to whole tissues,
biological fluids and proteins secreted in vitro, are open to
analysis by Western blotting
 In most cases, the cells are harvested, washed, and lysed
to release the target protein
 For best results, all these steps should be carried out on ice
 This will minimize proteolysis, dephosphorylation, and
denaturation, since all begin to occur once the cells are
disrupted

9.  The choice of extraction method depends primarily on the sample
and whether the analysis is targeting all the proteins in a cell or only
a component from a particular subcellular fraction
 The endogenous proteases may be liberated upon cell disruption and
may degrade the target molecule
 the sample should be protected during cell disruption and subsequent
purification by the use of protease inhibitors to avoid uncontrolled
protein losses
 Assorted detergents, salts, and buffers may be employed to
encourage lysis of cells and to solublize proteins
 Tissue preparation is often done at cold temperature to avoid
protein denaturing

10. GEL ELECTROPHORESIS
The proteins of the sample are seperated using gel
electrophoresis. Seperation of proteins may be by isoelectric
point molecular weight, electric charge, or a combination of
these factors
The principle involved is the difference in the
ELECTROPHORETIC MOBILITIES of different proteins
 Mix ingredients in the order shown above, ensuring no air
bubbles form.
 Pour the separating gel into glass plate assembly.
 Overlay gel with water to ensure a flat surface and to exclude
air.
 Leave to polymerize for ~ 20 minutes.
 Then prepare the stacking gel and pour on the running gel,
insert comb and leave for 20 min.

11. SAMPLE BUFFER
A sample of protein, is boiled in sample buffer
(at 95oC for 5 minutes) which contains:
 The β-mercaptoethanol reduces disulfide bonds
 SDS (Sodium dodecyl sulfate) disrupts protein secondary
and tertiary structure
 Glycerol to make samples sink into wells
 Bromophenol Blue dye to visualize samples

12.  The end result has two important features:
1. All proteins contain only primary structure and
2. All proteins have a large negative charge which means
they will all migrate towards the positive pole when
placed in an electric field.
 They migrate through a gel towards the positive pole
at a rate proportional to their linear size

13. Loading Samples & Running the gel
 Samples are loaded into
separate wells
 A protein marker is also
loaded
 Run for 30-40 minutes
 Running Buffer (pH 8.3)
contains
 Tris Base
 Glycine
 SDS

14. PROTEIN TRANSFER
 In order to make the proteins accesible to antibody
detection, they are moved from within the gel onto a
membrane made of nitrocellulose or polyvinylidine
difluoride(PVDF).
 The membrane is placed on top of the gel, and a stack of
filter papers placed on top of that. The entire stack is
placed in buffer solution which moves up the paper by
capillary action, bringing the proteins with it.
 Another method for transferring the proteins is called
electro blotting and uses an electric current to pull proteins
from the gel into PVDF or nitrocellulose membrane

15. BLOCKING
 Blocking is a very important step in the immunodetection
phase of Western blotting because it prevents non-specific
binding of antibody to the blotting membrane
 The most commonly used blocking solutions contain
bovine serum albumin 3-5% (BSA) or non-fat dried milk
in a solution of PBS (phosphate buffered saline) or TBS
(tris buffered saline)
 Often, a small amount of Tween 20 detergent is added to
blocking and washing solutions to reduce background
staining, and the buffer is known as PBST or TBST

16. ANTIBODY PROBING
 Once the protein samples are separated and transferred onto a
membrane, the protein of interest is detected and localized
using a specific antibody.
 The blot will be incubated in a dilute solution of antibody,
usually for a few hours at room temperature or overnight at
4°C.
 The antibody is diluted in wash buffer (PBST or TBST) or in
the blocking solution, the choice depends upon the antibody
 Since antibody preparations vary in their levels of purity and
specific binding properties, there will be differences in the level
of dilution required

17.  Usually non-labeled primary antibody directed against the
target protein.
 Wash the membrane several times in TBST while agitating, 5
minutes or more per wash, to remove residual primary antibody
 A species-specific, labeled secondary antibody directed against
the constant region of the primary antibody is then used
 The secondary antibody serves not only as a carrier of the label
but is also a mechanism to amplify the emitted signals, as many
secondary antibodies can theoretically bind simultaneously to
the primary antibody
 Secondary Ab is also diluted according to the manufacturer
recommendations and incubated for 1 hour at RT

18. DETECTION WITH SUBSTRATE
 The most common antibody label used in Western blots is
horseradish peroxidase (HRP), a small, stable enzyme
with high specificity and rapid turnover
 The signal is detected when HRP is exposed to a substrate
solution in the final step of the immunodetection
procedure
 Substrate solutions for Western blotting are chemical
reagents that are acted upon by the enzyme to yield a
signal that can be easily measured
 HRP label is typically detected with either colorimetric.

20. APPLICATIONS