I am running some pulldown experiments using plasma samples(click biotin linker on protein, then blot against Strep-HRP). We started the experiment with a known protein (Lane 2-5) then plasma samples (Lane6-9), lane 5 and 9 both incubated with alkyne-biotin. Although the signal is very small and on WB I need to overexpose to see it, but the bands on lane 9 is giving a bizarre signal. Could anyone help me understand the reason for this and how to solve it?
Please repeat the experiment and apply adequate amount of Antibody .primary/ secondary Antibody well. May be there was a problem while shaking or the amount of Antibody applied was less than required so, the PVDF membrane might not have bathed well in it. What happened in the 2nd picture is that when the exposure time was increased, the luminol in lane 9 got bleached. It does not indicate any proper band but only the bleaches.
Please repeat the experiment and apply adequate amount of Antibody and shake the box with the PVDF membrane well.
Thank you Anupriya, the antibody concentration is 1:50000, I have also tested 1: 10000(attached picture last two bands) Strep-HRP(20ml, completely cover the blot) but I think the result is similar only more intensed. I also prepared fresh antibody each time.
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