What is your method of detection? In my experience heart tissue may cause background issues when using fluorescent secondary antibodies. Never had any problems using chemiluminescent based detection.

Hi Yvette,

:) thanks for the info.. Yes we did longer blocking.. but now the target band masks. We tried to increase the protein loaded/well. It gives a huge nonspecific band with a very faint target band.

Cheers,

Susara

Blocking with BSA always gives sharp bands compared to skimmed milk. You can try blocking with 5-10% BSA in TBST for 1-2 hours.

Remember to filter the BSA after preparation.