What is the maximum amount of protein that could be loaded in WB? - (Oct/12/2006 )
Hi, all,
The protein I am using Wersten Blotting to detect is one of mitochondrial inner membrane protein (UCP2), since there is no a perfect antibody on the market, so the signal is very weak when I loaded the common ammount of sample proteins (30-40ug).
So if I want to increase the signal by increasing the amount of loading samples, what is the maximum amount I can use? I heard from someone else that too much of samples could lead to a false positive result, is that correct?
Does anyone has some experience on the WB of the same protein? Could you recommend a good commercial antibody?
Thanks for your kind reply
To much protein (> 50 ug) may increase your background. Others have attained good results in cell culture. See:
Bordone L, Motta MC, Picard F, Robinson A, Jhala US, et al. (2006) Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells. PLoS Biol 4(2): e31
The protein I am using Wersten Blotting to detect is one of mitochondrial inner membrane protein (UCP2), since there is no a perfect antibody on the market, so the signal is very weak when I loaded the common ammount of sample proteins (30-40ug).
So if I want to increase the signal by increasing the amount of loading samples, what is the maximum amount I can use? I heard from someone else that too much of samples could lead to a false positive result, is that correct?
Does anyone has some experience on the WB of the same protein? Could you recommend a good commercial antibody?
Thanks for your kind reply
The protein I am using Wersten Blotting to detect is one of mitochondrial inner membrane protein (UCP2), since there is no a perfect antibody on the market, so the signal is very weak when I loaded the common ammount of sample proteins (30-40ug).
So if I want to increase the signal by increasing the amount of loading samples, what is the maximum amount I can use? I heard from someone else that too much of samples could lead to a false positive result, is that correct?
Does anyone has some experience on the WB of the same protein? Could you recommend a good commercial antibody?
Thanks for your kind reply
to answer the question properly one has to know if you run your protein pure or in a mitochondrial lysat fraction; moreover, size of protein is to know
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