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: Understanding the Concept:
The question asks for the correct chronological sequence of the major steps in a Western blot, a technique used to detect a specific protein in a complex mixture.
Step 2: Detailed Explanation:
The logical workflow of a Western blot is as follows:
D. Protein denaturation in loading dye. First, the protein sample is prepared by boiling it in a loading buffer containing a detergent like SDS (sodium dodecyl sulfate). This denatures the proteins (unfolds them) and gives them a uniform negative charge.
A. Protein resolution by PAGE. The denatured protein mixture is loaded onto a polyacrylamide gel, and an electric current is applied. The proteins migrate through the gel and are separated based on their size. This is PolyAcrylamide Gel Electrophoresis (PAGE).
C. Transfer onto nitrocellulose membrane. After separation, the proteins are transferred (blotted) from the fragile gel onto a solid support, typically a nitrocellulose or PVDF membrane. This makes the proteins accessible for antibody binding.
B. Primary antibody binding. The membrane is then incubated with a primary antibody that is specific to the protein of interest. This antibody binds only to the target protein on the membrane. (This is followed by washing, secondary antibody binding, and detection, but primary antibody binding is the next key step listed).
Therefore, the correct sequence is D \(\rightarrow\) A \(\rightarrow\) C \(\rightarrow\) B.
Step 3: Final Answer:
The correct order of events is D, A, C, B.