SDS PAGE - some problems in results (Nov/20/2004 )

Hello everybody!!!!!
I am having some problems with my protein purification actually I am not able to see the band in my PAGE. I have tried all the different conc. of gel but still failed to get the bands so if anybody could tell me the reason for it and give me some suggestions! I think my protein is a low molecular weight protein with mol. wt. of 30 to 50kd so are ther any specific technique for their separations? please do tell me

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Hi there,

Are you running a positive control on your gel to check that your staining method works? what method do you use to stain your gel?

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Are you sure the protein has been experssed?

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Hi,

I think, you problem not because of the size of your protein. For range of 30-50 kDa normal Laemmlii technique should be fine! But some proteins can not be stain with Coomassie blue. Try Ponceau S, negative cupper or zinc staining, silver staining, etc. Or try to detect your protein with Western.

Alexei

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well how do u make sure that protein is there in the sample u r loading on the gel.

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hi there,
I am having protein in my sample. so what do u think is there any need of confirmation of its presence?

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hi
first of all thanks for ur reply. I have tried for silver staining with all different gel % but failed and i dont know about zinc or -ve copper stainig so please give me information about them and what is their specificity.
Thanks

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I am doing the enzyme activity of it and so I am very sure that the protein is expressed.

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I do use marker for check. it is coming but not my protein either it is not at all coming in the gel or what is the problem i am not getting it I have tried for sds-page and native page for all different gel % but still failed!!!! so please give me if any suggestions or ideas are there related to this aspect

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