Gel is 10%, used 1% BSA for blocking in PBST. Primary antibody dilution 1:1000 for three hours at room temp. and secondary dilution 1:5000 for 1 hour at room temp.

In my expirience once I had a membrane which gave such background. It does not depend on transfer or bloking procedure, just try another membrane.

I did not perform protein precipitation by TCA or ammonim sulphate. I transferred the protein for overnight at 4 oC and 30V. the buffer used for blocking is 1% BSA in PBST.