2. WESTERN BLOTTING:
A western blotting is a laboratory method used to detect
specific protein molecules from among a mixture of proteins.
This mixture can include all of the proteins associated with a
particular tissue or cell type.
Western blots can also be used to evaluate the size of a
protein of interest, and to measure the amount of protein
expression.
Also called Protein blotting.
Western blotting is also known as immuno blotting because it
uses antibodies to detect the protein.
4. Principle :
• It is based on the principle of immuno chromatography
where proteins are separated into polyacrylamide gel
according to their molecular weight.
• The proteins thus separated are then transferred onto
nitrocellulose membrane and are detected using specific
primary antibody and secondary enzyme labelled
antibody which in the end we will get colored product.
• The colour indicate the presence of protein of interest.
5. Procedure :
1-Sample preparation
2-Gel Electrophoresis
3-Blotting (Transfer)
4-Blocking
5-Treatment with primary antibody
6-Treatment with secondary antibody
7-Detection
6. 1. Step -1 :Sample preparation
First step in the sample preparation is isolating the
protein from a sample.Usually proteins are purified
from cells.
Next the protein concentration is determined. Sample
buffer commonly Leammli buffer which contains
Sodium dodecyl Sulfate (SDS) and beta-
mercaptoethanod(BME) is added to the protein
suspension.
The sample buffer is heated to near boilling, which
denatures the protein and allows the SDS to bind the
protein ,which make the protein unfold in to linear
chains and coats than with a negative charge.
7.
8. • Step-2 Gel Electrophoresis
The sample is loaded in the polyacrylamide gel.
The protein are separated on the basis of electric charge,
molecular weight.
Small size protein move faster than large size protein.
Protein are negatively charge, so they move toward
positive (anode) pole as electric current is applied.
9. Step-3 Blotting (Transfer)
The nitrocellulose membrane is placed on the gel. The
separated protein from gel get transferred to the
nitrocellulose membrane by capillary blotting. This type of
blotting is time consuming and may take 1-2 days.
For fast and more efficient transfer of desired protein from
the gel to nitrocellulose membrane electro blotting can used.
In electro blotting nitrocellulose membrane and gel is
sandwich between filter paper and sponge(fiber pad).
And electric current passed through the gel causing transfer
of protein to membrane.
10.
11.
12. Step-4 Blocking
The membrane has ability to bind to protein.
In this case both target and antibodies are proteins and so
there could be so unwanted binding.
Blocking of non-specific binding is achieved by placing the
membrane in a dilute solution of protein typically 3-5%
bovine serum albumin (BSA) or non fat dry milk in tri-
buffered saline.
The protein in the dilute solution attaches to membrane in all
places where the target proteins have not attached.
Thus, when the antibody added, there is no room on the
membrane for it to attach other than on the binding sites of
the specific target protein.
13. Step-5 Treatment with Primary
antibody
After blocking, membrane is then incubated with primary
antibody, which specifically binds to the target protein.
Non-bound antibodies are washed off the membrane.
14. Step-6 Treatment with Secondary
antibody
Secondary antibody specifically recognized and binds to the
primary antibody.
Secondary antibody is enzyme labelled. (Alkaline phosphate
or Horseradish peroxidase (HRP) .
15. Step-7 Detection
A substance reacts with enzyme that is bound to secondary
antibody to generate colour or light, which allows it to easily
detect and imaged.
16.
17. Application :
To determinethe size and amount of protein in given sample.
Disease diagnosis : detects antibody against virus or bacteria
in serum.
Western blotting technique is confirmatory test for HIV. It
detects anti HIV antibody in patient's serum.
Useful to detect defective protein For eg Prions disease.
