Question

Which of the following would work best in Western blot if you could not detect low amounts of protein in the SDS-PAGE?

1. Use HRP-Ni2+ conjugate for the bovine LDH

2. Use a primary antibody for barracuda and the anti-LDH human secondary antibody

3. Use a primary antibody with a Nickel binding site to recognize a primary antibody to barracuda DNA

4. Use HRP-Ni2+ conjugate with a His tagged LDH

5. We would not be able to detect protein in western if it was not seen in SDS-PAGE

Answer 1

We would not be able to detect protein in western if it was not seen in SDS-PAGE

SDS PAGE is the initial step in western blotting technique in which the SDS PAGE bands will be transferred to nitrocellulose membrane and will be detected using primary and secondary antibody that bds with protein of interest and flouresce upon addition of substrates. If the SDS itself does not show any band, it is difficult to find in western blotting. We need to prepare higher amount of protein