The blocking buffer I am using is 1% BSA in PBS. I am using immunocap antibody dilutions. Regarding capture and detection I am using 2ug/ml and 1ug/ml respectively from the stock of 0.5mg/ml.
Thanks a lot sister. The protein i want to detect is casein, alpha lactalbumin and beta lactoglobulin, when i do milk specific IgG4 ELISA. Now i am doing total IgG4 ELISA. Please also suggest me about the blocking step. Thanks a lot.
You could try increasing the BSA to 3% and blocking for a longer period of time. When we block with BSA we typically do it overnight. You can also tinker around with your ab dilutions, they may be too high in concentration.
Hello Joyce, thanks for your reply. I have tried overnight incubation also. but it did not changed. I have also tried different blocking reagent (BD, Biolegend, R & D, our own lab reagent- 1% BSA+TRIS+NaCl). we are also using the recommended antibody concentration that the provider suggest. Anymore suggestions? Thanks again.
Hi Tanvir, I don't have any more suggestions but I have used concentrations recommended by the provider and I still get non-specific binding. I would still recommend trying higher dilutions on the antibodies. Some of my antibodies recommended I do a 1000 fold dilution and I've had to do 5000 and even 10,000 fold dilutions.
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