Question

You are studying a protein of 100 kilodaltons (kd). You found a way to cleave this protein at a single site¼ of the length from the N-terminus. You have raised a monoclonal antibody that binds to the N-terminus of protein X.

You run on an SDS-PAGE a sample of intact protein X (on lane 1) and a sample of protein X that has been cut at that single site (lane 2). If you do a Western Blot analysis with the monoclonal antibody what will be the size of protein band(s) observed in lanes 1 and 2?
1) Lane 1 = 100 kd Lane 2 = 25 + 75 kd
2) Lane 1 = 25 + 100 kd Lane 2 = 25 kd
3) Lane 1 = 75 kd Lane 2 = 75 kd
4) Lane 1 = 100 kd Lane 2 = 25 kd
5) Lane 1 = 25 kd Lane 2 = 25 kd

Answer 1

Option 4.

In the western blot analysis, lane 1 would display a 100 kd band (intact protein), and lane 2 would display a 25 kd band (N-terminal fragment), making option 4 the correct answer.

Step-by-step explanation:

In western blot analysis, you would expect to see the following results given the described scenario:

1. Lane 1 would show a single band at 100 kilodaltons (kd) since the protein is intact and the monoclonal antibody will detect the N-terminus of the full-length protein.
2. Lane 2 would show a single band at 25 kilodaltons (kd) because the protein has been cleaved ¼ from the N-terminus. Hence, the antibody would only detect the N-terminal fragment, which is ¼ of the full length, equating to 25 kd out of the initial 100 kd.

A band corresponding to the 75 kd fragment would not be detected in lane 2 since the antibody is specific to the N-terminus of the protein, which is not present in the 75 kd fragment. Therefore, the correct option in the final answer is option 4.