Dot Blot - Protein not adhering to membrane - (Sep/26/2011 )

Hi, we are having problems with a dot blot for insulin. We have tried both nitrocellulose and PVDF.

When we used nitrocellulose we got zippo for the insulin (but the control BSA was good). Afterwards we did a stain for protein and it seemed the insulin washed off.

When we used PVDF we got very faint results for insulin (but once again the control BSA was good).

Any suggestions on how to make the insulin more attractive to the membrane?

-Serena

What is the pore size of your membrane? You might want to try 0.2µm pore size if you are not already doing so.

How do you do your dot-blotting?