Question

When developing a western blot what two factors influence the intensity of the band?

Answer 1

Western blot is the procedure to analyse production of proteins in a cell in any environment. The band size may be affacted by many factors like -

1. Membrane used for blotting - PVDF and Nitrocellulose are the membranes used for blotting. These membranes binds with protein by hydrophobic forces as well as ionic interactions. These binds mostly non specifically but affinity may not always be same. PVDF membrane are less fragile but bind more non specifically but Nitrocellulose membrane are more fragile and binds less specifically.

2. Primary and secindry antibody - use of lebelled secondry antibodies enhance the band strength because more secondary antibodies bind with primary antibodies bind to proteins.

3. Protein size and concentration - Transfer efficency is influenced by size of protein and molecular weight. Protein less tha 60 KDa are transferred easily and also the concentration if more than band will be darker and large.