Hello,

Sometimes PVDF membranes need to be activated by methanol, I do not know if this is your case. Have you ever stain your PVDF membrane too? You can check if your proteins are transfering to your PVDF membrane by a Ponceau red on your membrane

Background is simply because of non specific binding of antibodies, use BSA (1%) along with antibodies.

Reduce exposure time also go through western blot technique by tahrin north american j med science 2012 ,4, 429-34

Are you using a positive control for your antigen? Are your antibody concentrations optimised?

yes methanol should be used and PVDF membranes dipped in that before putting for transfer,and for background-use 5% in 1x tbst skimmed milk for blocking either overnight or 1 hr before primary antibody treatment.it helps to reduce background.

try to use thinner gel for efficient transfer and optimize transfer time and buffer. I hope it may help.

Thank you all for your suggestions.

I do activate the PVDF membrane in methanol for 5 minutes and then let it equilibrate in the transfer buffer for 15-20 minutes before I initiate the transfer process.

I have also tried staining the membrane with Ponceau Red. Although at this stage the bands which I can see on the membrane are very faint (I will upload a picture the next time I perform this)

Pete, I have not been using positive controls. I will try that and let you know.

Mahesh, Meeta, Shazia and Hemant, I will try out your suggestions and get back to you.

Thanks again.

Have you checked your antibodies?