control is positive, sample is negtive - (Jun/17/2007 )
Dear all:
I am trying to detect the expression in wheat leaves of a soluble mitochondrial protein (it is a subunit of the ATP synthase, I referred to it as P here for convenience) these days but failed. The primary antibody was produced by immuning rabbits with Tag-P fusion protein that expressed from E.Coli with the pET-32a vector. The primary antibody seems work well since it can bind to the Tag-P fusion protein in a western blot assay. However, when I used it to hybridize with the total proteins extracted from wheat leaves in a western blot experiment, no signal appeared, with only the lane loaded with the Tag-P fusion protein control positive. My question is that (1) Is this due to the primary antibody only can bind with Tag rather than P? or (2) Is my method in extracting wheat leaf proteins inappropriate? I isolated the proteins with 10mM Tris by grinding the leaves. My target protein is a soluble mitochondrial protein.
Thanks a lot.
Yours, paul
I'm not entirely sure that simple grinding will disrupt the mitochondria. I'd be tempted to try something else, like freeze-thawing after the grinding step.
Your other question, about the effect of the Tag on the detection is a good one. Can you isolate some P without the Tag? and see if the antibody picks it up (or the Tag, for that matter)?
Your other question, about the effect of the Tag on the detection is a good one. Can you isolate some P without the Tag? and see if the antibody picks it up (or the Tag, for that matter)?
Your Ab is rabbit, so polyclonal, so should recognize more than one epitope. If your P is not too small compared to Tag, you should be fine (cross your fingers). To make sure that you disrupt the mitochondria properly, could you find a positive control, like a known mitochondrial protein with available Ab and WB parallel with your Ab? If that works, and yours not, then worried about Ab.
If the Ab recognize the Tag, that is easy to solve, but if it recognize an epitope that is part Tag-part P, then big trouble. Have to try to get P without Tag, pure Tag and Tag-P to try and see if it is really the case.
thank to both replies!