I have found same signal from control and sample in DOT-BLOT Assay. What is the precaution needed for optimization of this?

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Wolfgang Schechinger

How are you blocking the membrane?

Sourav Kumar

I have used 1% BSA in TBS as a blocking solution.

Try adding 0.1 or 0.2% m/v Tween 20 to all solutions but what you are using for coating. Also do checkerboard dilutions of antigen and antibody and determine the optimal dilution of the anti rabbit HRP first (how much may you dilute it before you see background?).

If you have access to ELISA-plates and a microplate reader, this approach might give you semi-quantitative data.