#blotting

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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Blotting Techniques

Which blot uses RNA as the target molecule?

Northern blotting is historically named by analogy to Southern blotting and is dedicated to RNA target analysis. Total cellular RNA or poly(A) enriched mRNA is denatured with formaldehyde or glyoxal to eliminate secondary structures and separated on agarose gel according to length. After capillary or electro-transfer to positively charged nylon membrane and UV crosslinking, transcripts are hybridized with labeled antisense DNA or RNA probes. Autoradiographic signal indicates transcript size and abundance. Although largely supplanted by quantitative RT-PCR and RNA sequencing for sensitivity, Northern remains gold standard for validating transcript integrity and isoform size without amplification bias.

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

Which technique separates proteins based on size before blotting?

Sodium dodecyl sulfate polyacrylamide gel electrophoresis provides indispensable size-based separation before Western blotting. SDS is an anionic detergent binding at approximately 1.4 grams per gram of protein, masking intrinsic charge and unfolding polypeptides into rod-like structures. Inclusion of reducing agents like dithiothreitol cleaves disulfide bridges. Migration through polyacrylamide sieving matrix then depends solely on polypeptide length logarithmically. This allows precise molecular weight estimation and resolution of isoforms. Techniques such as RT-PCR amplify nucleic acids, electroporation introduces DNA into cells, and ELISA detects without size resolution, none replicate SDS-PAGE function.

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

Which membrane offers highest durability and is suitable for reprobing?

Polyvinylidene difluoride, marketed as PVDF, is a fluoropolymer membrane distinguished by exceptional mechanical toughness, chemical inertness, and thermal stability. Unlike nitrocellulose which becomes brittle upon drying and cannot endure harsh stripping buffers, PVDF remains flexible and intact through exposure to SDS, beta-mercaptoethanol, heat, and low pH glycine treatments. Hydrophobic and dipole interactions confer binding capacity of approximately 170-200 micrograms per square centimeter, much higher than nitrocellulose. Methanol activation is prerequisite. Durability permits sequential probing for multiple antigens, loading controls, and phosphorylated forms from same precious lysate.

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 type of bond forms between DNA and positively charged nylon membrane?

Positively charged nylon membranes contain quaternary ammonium groups that initially attract negatively charged phosphate backbone of DNA through electrostatic interaction during Southern or Northern transfer. For permanent immobilization, membrane is exposed to ultraviolet irradiation at 254 nm. Photochemical reaction creates covalent bonds between thymine residues of DNA and amine groups of nylon matrix. This covalent fixation withstands high stringency hybridization, high temperature washing, and repeated stripping cycles. By contrast, nitrocellulose relies solely on hydrophobic interactions, and neutral nylon without crosslinking shows lower retention, explaining why UV-induced covalent linkage is standard for nylon membranes.

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

Which blotting method is used to detect DNA?

Southern blotting, invented by Edwin Southern in 1975, remains the definitive method for detection of specific DNA sequences. Genomic DNA is fragmented with restriction endonucleases, resolved by agarose gel electrophoresis according to size, denatured under alkaline conditions to single strands, and capillary transferred to nitrocellulose or nylon membrane. Immobilized fragments are hybridized with radioactively or non-radioactively labeled complementary probes. Detection reveals fragment length polymorphism, enabling genotyping, DNA fingerprinting, transgene confirmation, and analysis of gene rearrangements with high specificity and reproducibility.

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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