Question

Arrange the following events in Western Blotting experiment in correct order:
A. Protein resolution by PAGE
B. Primary antibody binding
C. Transfer onto nitrocellulose membrane
D. Protein denaturation in loading dye.
Choose the correct answer from the options given below :

• A. A, C, B, D
• B. D, A, C, B
• C. B, A, D, C
• D. C, B, D, A

Hint:

For blotting techniques (Southern, Northern, Western), remember the general order: {Separate} (on a gel), {Transfer} (to a membrane), and then {Probe} (with DNA, RNA, or antibody). The initial sample preparation (like denaturation) must come first.

Answer 1

The Correct Option is B

Step 1: Concept Overview:
The question requires sequencing the major steps of a Western blot, a technique for detecting specific proteins in a sample.
Step 2: Step-by-Step Breakdown:
The Western blot procedure involves these key steps:
D. Protein denaturation with loading dye. The protein sample is prepared by boiling it in a loading buffer containing SDS. This denatures the proteins and imparts a uniform negative charge.
A. Protein separation via PAGE. The denatured protein mixture is subjected to polyacrylamide gel electrophoresis (PAGE). Proteins migrate through the gel and are separated by size.
C. Transfer to nitrocellulose membrane. The separated proteins are transferred from the gel to a nitrocellulose or PVDF membrane. This transfer allows for antibody binding.
B. Primary antibody binding. The membrane is incubated with a primary antibody specific to the target protein, which binds to the protein on the membrane. (Subsequent steps include washing, secondary antibody binding, and detection; however, primary antibody binding is the next listed step).
Therefore, the correct chronological order is D \(\rightarrow\) A \(\rightarrow\) C \(\rightarrow\) B.
Step 3: Conclusion:
The correct sequence is D, A, C, B.