I did a western blot with a PVDF membrane. Because I only have 5 samples, I have thought of using 5 ml (1:1000 antibody dilution) of antibodies (Ni-NTA AP Conjugate). Unfortunately, I have detected a signal from 1 lane only.
I have previously used 10 ml and it worked perfectly. However, to save items, I halved the amount. Can it be that the reason for the absence of bands is due to the insufficient amount of antibodies?
Thanks.
Thank you for your response. I have checked the transfer via dyeing my gel with Coomassie blue. As it seems, the proteins have successfully transferred. I am using QIAexpressionist Ni-NTA AP Conjugate.
Ernest, as long as the blot is submerged and the antibody has access to all parts of the blot, the volume is not the issue. Each antibody is different from the next, so its optimal dilution needs to be determined. Since you checked transfer, there are no issues there. You detect a band in one lane only, that might be your positive control? If so, the antibody works and the problem may be the samples in the other lanes.
Thank you very much everyone for your helpful responses Mr. Haque and Mr. Voskuil.
@Mr. Haque I am using 3% BSA for blocking incubated with the membrane for 1 hour. I do not use a secondary antibody, as the primary antibody is already conjugated with the enzyme for detection. Thank you.
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