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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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At wits end with no signal
Posted by: Heatherbee (IP Hidden, New member, 3)
Date: April 19, 2005 11:10AM
I have been trying unsucessfully for weeks to get a signal for A1 protein off of samples that I have run for 6 other antibodies by western with no problems at all. After getting free antibody replacements from the company I have tried EVERYTHING I can think of (believe me when I say everything-all kinds of membrane, checked transfer, different block proteins, sodium azide, incubation times varying in length, concentration and agitation, even different antibodies for the same protein!) and I still get no bands of the appropriate size...not even close. I have struggled for a while to get any bands at all - now I do, but they are not even close to the size I need (~20 KDa). I am at my wits end, as I do not have much sample to work with and I need the results to complete a study. I have tried diffferent samples I have a lot of (to conserve the important ones) and incubated in primary antibody overnight and am hoping this may help... I'd like to move onwards but am stuck with this protein work....has anyone on this forum used A1 antibody in human protein before? Is there a need to load unusually large amounts of protein to get a signal? Should I stand on my head and rub my belly at the same time?? :)
Any advice
Re: At wits end with no signal
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 20, 2005 10:51AM
Hi,
I think I know the feeling. ALthough I do not have experience with this type of protein, aAre these bands you see are isoforms of Protein A of the protein or just a result of cross reactivity? also do you know if this protein is post-translationaly modified (may be by phosphorylation) and may be this is why you getting several bands on your blot. If this protein is heavly phosphorylated, I would suggest to treat your protein lysate with alkaline phophatase before running it on the gel, may be this will provide you will less bands and hopefully 20 kDa. Another possibility to explore further if this protein is membrane or cytoskelton associated and thus you need to treat your cells with a strong denaturing detergnet such as SDS to ensure complete solubility of protein sample. I hope these notes will be of hel to you, Good Luck!
Re: At wits end with no signal
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 20, 2005 01:48PM
Hi,
I also have not worked with your protein, although I think we've all had this problem before. Are the bands you see of lower apparent molecular weight than the full length protein of interest? If so it is possible that what you are seeing is the result of proteolytic breakdown of the antigen, which can occur if the samples are stored for prolonged periods of time or if the proteins become fractionated during homogenization. If this is the case I do not know if there is anything you can due to restore the samples once this breakdown has occurred. You may want to consider adding a protease inhibitor such as PMSF, pepstatin or leupeptin to your samples in the future before storage or homogenization. Good luck!
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