The document discusses Western blotting, which is a technique used to detect specific proteins in a biological sample. It involves separating proteins by size using gel electrophoresis, transferring them to a membrane, and using antibodies to detect the target protein. The key steps are separating proteins by SDS-PAGE, transferring them to a membrane using blotting, and detecting the target protein with primary and secondary antibodies to visualize protein bands. Western blotting allows identification and quantification of proteins and is widely used in research and clinical diagnosis.

1. Bishal Panth
Msc. Biotechnology
Second Semester
National College
1
Blotting
Western

2. Content
Introduction on blotting
Types of Blotting
Western Blotting ( Principle, procedure)
Reference
2
Bishal Panth || Msc. Biotech

3. 3
• Blotting techniques are molecular methods used to identify and
measure specific DNA, RNA and protein in complex biological
mixtures.
• Visualization of specific DNA , RNA & protein among many
thousands of contaminating molecules.
• It is the technique for transferring DNA , RNA and proteins onto a
carrier so they can be separated, and often follows the use of a gel
electrophoresis.
Introduction on blotting
Bishal Panth || Msc. Biotech

4. 4 Types of blotting
Bishal Panth || Msc. Biotech
Blotting
Southern
Blotting
Northern
Blotting
Western
Blotting
DNA RNA Protein

5. 5  Western blotting
Bishal Panth || Msc. Biotech
• Western blotting is a widely used analytical technique in molecular biology to
detect specific protein in a complex biological sample or extract.
• It is an immunoblotting (protein detection) technique using the separation
power of SDS PAGE into an absorbent membrane to assess the presence,
amount and molecular- weight of proteins in cellular or tissue extracts using
antibodies.
• It works on the principle of gel electrophoresis. Proteins are separated based
on their size on polyacrylamide gel
• The method is characterized by transferring the protein, which was run on a
gel by electrophoresis, onto a nitrocellulose membrane. This makes the
protein stable on the membrane so that several methods including methods of
detection and quantity of the protein content can be detected.

6. 6
Bishal Panth || Msc. Biotech
 Western blotting- Principle
Western blot is performed by using polypropylene gel electrophoresis. SDS-PAGE
allows protein samples to be separated and transferred to a solid support, such as
nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane. The solid support
called blot absorb the protein and keep its biological activity unchanged and is treated
with a protein solution to block the hydrophobic binding site on the membrane. The
membrane is treated with the antibody (primary) of the target proteins. Only the
proteins to be studied can specifically bind to the primary antibody to form an antigen-
antibody complex. The primary antibody-treated membranes are treated with a labeled
secondary antibody after washing. After treatment, the labeled secondary antibody that
binds to the primary antibody forms an antibody complex that can indicate the
location of the primary antibody, both the location of the protein being studied.

7. 7  Western blotting- Procedure
Bishal Panth || Msc. Biotech
The technique consists of three major processes:
1.Separation of proteins by size (Electrophoresis).
2.Transfer to a solid support (Blotting)
3.Marking target protein using a proper primary and
secondary antibody to visualize (Detection).

8. 8
Bishal Panth || Msc. Biotech
Electrophoresis is used to separate proteins
according to their electrophoretic mobility which
depends on the charge, size of protein molecule,
and structure of the proteins. Proteins are moved
within the gel onto a membrane made of
Nitrocellulose (NC) or Polyvinylidene difluoride
(PVDF). The proteins combine with nitrocellulose
membrane based on hydrophobic interaction.
Fig: SDS Electrophoresis || Source : Research gate
 Western blotting- Procedure

9. 9
Bishal Panth || Msc. Biotech
 Western blotting- Procedure
Western blot uses two types of agarose gel: Stacking gel that is used
for concentrate all proteins in one band and Separating gel that
allows for separating proteins according to their molecular weight.
Fig: SDS Electrophoresis || Source : Proteomics.com

10. Bishal Panth || Msc. Biotech
10
Fig: Blotting technique || Source : Proteomics.com
 Western blotting- Procedure
• A foam sponge is taken and laid on the backside, over
which goes the filter paper. These should be placed to
ensure that both of them are wet and slightly submerged.
• The gel is taken out from the tank and placed on the wet
filter paper.
• The nitrocellulose membrane is wet with the transfer
buffer and is placed on top of the gel in a way that there
are no bubbles between the gel and the membrane.

11. Bishal Panth || Msc. Biotech
11
Fig : Schematic of electrophoretic protein separation in a polyacrylamide gel || Source : bio-rad.com
 Western blotting- Procedure
For detection of the proteins, primary antibody and enzyme-conjugated
secondary antibody are used. In addition of substrate, a substrate reacts with
the enzyme that is bound to the secondary antibody to generate colored
substance, namely, visible protein bands.
• Protein bands appeared after protein transfer with buffer in a
membrane gives the data for the sample used.
• The protein levels can be evaluated by spectrophotometry.
• It is used in the clinical diagnosis of different diseases. The
confirmatory test for HIV involves a western blot by detecting
anti-HIV antibodies in the serum.
• used to quantify proteins and other gene products in gene
expression studies.
• It is also used for the analysis of different biomarkers like
growth factors, cytokines, and hormones.

12.  Western blotting
Bishal Panth || Msc. Biotech
12
Fig: Process of Western Blot || Created with BioRender.com || Source : Microbes Notes

13.  Reference
i. Kurien BT, Scofield RH. Western blotting: an introduction. Methods Mol Biol.
2015;1312:17-30. doi: 10.1007/978-1-4939-2694-7_5. PMID: 26043986; PMCID:
PMC7304528.
ii. Hnasko TS, Hnasko RM. The Western Blot. Methods Mol Biol. 2015;1318:87-96. doi:
10.1007/978-1-4939-2742-5_9. PMID: 26160567.
iii. Mahmood, Tahrin, and Ping-Chang Yang. “Western blot: technique, theory, and trouble
shooting.” North American journal of medical sciences vol. 4,9 (2012): 429-34.
doi:10.4103/1947-2714.100998
iv. Ghosh, Rajeshwary et al. “The necessity of and strategies for improving confidence in the
accuracy of western blots.” Expert review of proteomics vol. 11,5 (2014): 549-60.
doi:10.1586/14789450.2014.939635
Bishal Panth || Msc. Biotech
13

14. Bishal Panth || Msc. Biotech
14