#immunoblotting

Immunological Applications

Immunoblotting is also known as:

Immunoblotting approach later termed Western blot combines size based separation with specific antibody detection, becoming gold standard for protein confirmation. Methodology introduced by Harry Towbin and Neal Burnette 1979: sodium dodecyl sulfate anionic detergent disrupts secondary and tertiary structure coating polypeptide backbone with uniform negative charge ratio approximately 1.4 gram SDS per gram protein, allowing electrophoretic mobility in polyacrylamide gel to correlate inversely with logarithm of molecular weight separating proteins from 10 to 300 kDa according to pore size determined by acrylamide percentage and bis-acrylamide crosslinking. Proteins then transferred electrophoretically from gel to nitrocellulose or polyvinylidene difluoride PVDF membrane via wet or semi-dry blotting applying electric field 100 volts for one hour preserving spatial pattern, membrane with high protein binding capacity via hydrophobic interaction. Membrane blocked with nonfat dry milk five percent or bovine serum albumin three percent containing Tween-20 to prevent nonspecific adsorption of antibodies. This mechanistic insight supports diagnostic and therapeutic applications while reinforcing core immunological and cell biology principles taught in advanced curricula.

Ref: Towbin et al PNAS 1979 76:4350 Western blot SDS-PAGE PVDF; NCBI Bookshelf immunoblotting Towbin transfer.

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Immunological Applications

Immunoblotting is also known as:

Western blotting immunoblotting combines SDS-PAGE separation proteins molecular weight antibody detection achieving high specificity confirmatory test protein expression. Lysate preparation RIPA buffer detergents 0.1 percent SDS anionic 1 percent Triton X100 nonionic 0.5 percent deoxycholate salts 150 mM NaCl protease inhibitors PMSF aprotinin leupeptin preventing degradation sonication shearing DNA. Proteins denatured 95C 5 min Laemmli sample buffer SDS conferring uniform negative charge mass ratio 1.4 g/g beta-mercaptoethanol 2 percent reducing disulfides glycerol density bromophenol blue tracking. Loaded discontinuous polyacrylamide gradient 4-20 percent separating gel pH 8.8 stacking pH 6.8 electrophoresis 100V where proteins migrate inversely log MW separating 10-250 kDa. Proteins electrotransferred nitrocellulose 0.45 micron PVDF 0.2 micron hydrophobic activated methanol via semi-dry tank wet transfer electric field 100V 1h 4C preserving spatial pattern facilitating immunodetection. Membrane blocked 5 percent nonfat milk TBS Tween blocking nonspecific adsorption primary antibody rabbit mouse specific epitope p53 DO1 beta-actin AC15 HIV gp120 incubated overnight 1:1000 followed washes HRP-conjugated secondary goat anti-rabbit mouse recognizing Fc. ECL substrate luminol oxidized HRP H2O2 generating photons captured X-ray film CCD camera band intensity densitometry quantified antigen amount. Name western coined Burnette 1981 analogous Southern Edwin Southern DNA

Ref: Towbin Staehelin Gordon 1979 Western Blot PNAS Protocol; Burnette 1981 Western Naming Analogy Southern Northern; Janeway Immunoblotting Technique Applications Chapter 3.

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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

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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