#Western blotting

Electrophoresis

What does Western blotting detect?

Blotting techniques are classified by target macromolecule: Southern blotting for DNA fragments, Northern for RNA transcripts, and Western for proteins, also called immunoblotting. Western blotting involves electrophoretic transfer of resolved proteins from polyacrylamide gel onto nitrocellulose or PVDF membrane, followed by blocking to prevent nonspecific binding and incubation with highly specific primary antibodies and enzyme-linked secondary antibodies for chemiluminescent or colorimetric detection. This identifies molecular weight, abundance and modifications of specific proteins. Lipids, DNA and sugars require thin-layer chromatography, Southern blotting or periodic acid-Schiff staining, not immunoblotting.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Blotting Techniques

A labeled antibody is required in:

Western blotting, synonymously called immunoblotting, relies fundamentally on specific antigen-antibody interaction for protein identification and quantification. Workflow involves SDS-PAGE separation, electrotransfer to membrane, blocking, incubation with unlabeled primary antibody against epitope, followed by labeled secondary antibody conjugated to horseradish peroxidase, alkaline phosphatase, fluorophore, or bioluminescent enzyme. Signal originates from label attached to antibody. Polymerase chain reaction amplifies DNA using oligonucleotide primers and does not involve antibodies. Southern and Northern blotting detect nucleic acids through labeled complementary probes, not immunoglobulins, highlighting unique requirement for labeled antibody in immunoblotting.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Blotting Techniques

What is the role of stripping and reprobing?

Stripping and reprobing constitutes essential procedure to reuse same membrane for detecting multiple different proteins. After initial detection, membrane is incubated in buffers containing SDS, beta-mercaptoethanol or dithiothreitol, and glycine at low pH or elevated temperature to dissociate and remove bound primary and secondary antibody complexes, while target proteins remain immobilized due to strong hydrophobic interactions with PVDF or nitrocellulose. Subsequent reprobing with antibody against different antigen, such as housekeeping protein beta-actin or phosphorylated isoform, allows normalization, conserves limited clinical or fractionated samples, and reduces inter-gel variability.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Blotting Techniques

A typical blocking agent used in Western blotting is:

Bovine serum albumin, a 66 kDa abundant plasma protein, is widely employed as blocking agent and antibody diluent in Western blotting and other immunoassays. At working concentration of 3 to 5 percent, BSA efficiently coats unoccupied membrane sites on PVDF or nitrocellulose without cross-reacting with most anti-mammalian antibodies, unlike milk which contains biotin and immunoglobulins that may interfere with certain detection systems. It provides gentle proteinaceous barrier, stabilizes diluted primary and secondary antibodies, reduces nonspecific electrostatic interactions, and maintains epitope accessibility, whereas Triton X-100 is detergent for permeabilization, SDS is denaturant, and TMB is substrate, not blocker.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Blotting Techniques

Blocking agents in Western blotting are used to:

Membranes used for Western blotting, whether nitrocellulose or PVDF, possess high protein binding capacity and inevitably expose vacant hydrophobic surfaces after antigen transfer. Without intervention, primary antibodies and enzyme-linked secondary antibodies adsorb directly to these empty sites, producing high background and obscuring specific bands. Blocking step saturates unoccupied area with inert proteins such as non-fat dry milk, bovine serum albumin, casein, or irrelevant immunoglobulins, often supplemented with non-ionic detergent Tween-20. This prevents nonspecific antibody capture, enhances signal-to-noise ratio, improves specificity, and enables detection of low-abundance antigens with quantitative reliability.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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