Hello, I have been performing Western Blot and I ran into this problem. My protein should have 2 bands, one with a higher kDa (precursor protein) and another with a lower kDa (mature protein). However, after primary antibody incubation at 4 degrees overnight and subsequent secondary antibody incubation at RT for 1hr, only the band with a lower kDa is visible. I have cut the membrane well above the kDa of the precursor protein.
My samples are animal samples. To me it seems that without precursors, there cannot be products. Has anyone experienced a Western Blot having no precursor molecule but only the mature molecules? Or does anyone have a thought to why this happens?
Please correct me if I am wrong -- any feedback would be much appreciated! Thank you.
Sure! My question is, my protein should have 2 bands, one with a higher kDa (precursor protein) and another with a lower kDa (mature protein). However, after primary antibody incubation at 4 degrees overnight and subsequent secondary antibody incubation at RT for 1hr, only the band with a lower kDa is visible.
Thanks for taking the time to read my question! I'll also edit it to make it clearer.
Does silver staining confirm you have some product at the precursor kDa, even if it’s just a smear?
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