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strange band in coomassie blue staining


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#1 szang

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Posted 16 January 2010 - 10:45 AM

Hi everyone,

Yesterday i stained my SDS-PAGE gel using coomassie blue, i find there is a band about 70kDa in every lane including sds loading dye lane without sample.

Does it show my sds loading dye is contaminated? i always use the same loading dye. or sds loading dye can be stained by coomassie blue?

Does anyone have this experience, please tell me

#2 1324409

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Posted 16 January 2010 - 03:45 PM

Hi,
What is your sample and how did u prepare it? A purified protein or cell lysate or...? 70kDa protein is likely to be a heat shock protein which might sometimes come along with the protein of interest being purified from bacteria. however if u see it even without sample, then the best explanation would be the one u did as far as I can imagine. I would simply take a fresh loading dye, which u r sure is not contaminated and see whether u still see this protein or not. U should not see it in the lane without sample since comassie only binds to proteins and nothing else that is present in loading dye. U might still see this protein in your sample lanes since it might be a common bacterial heat shock protein, which definitely comes along with Nickel beads. Keep me updated please.

Cheers,
Volkan

#3 szang

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Posted 17 January 2010 - 04:30 PM

Hi,
What is your sample and how did u prepare it? A purified protein or cell lysate or...? 70kDa protein is likely to be a heat shock protein which might sometimes come along with the protein of interest being purified from bacteria. however if u see it even without sample, then the best explanation would be the one u did as far as I can imagine. I would simply take a fresh loading dye, which u r sure is not contaminated and see whether u still see this protein or not. U should not see it in the lane without sample since comassie only binds to proteins and nothing else that is present in loading dye. U might still see this protein in your sample lanes since it might be a common bacterial heat shock protein, which definitely comes along with Nickel beads. Keep me updated please.

Cheers,
Volkan


Thank you very much. I will run another gel and load new loading dye and my old loading dye to see what happen.

#4 mdfenko

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Posted 19 January 2010 - 07:44 PM

you may be seeing an artifact that appears as the reducing agent ages. it is from keratins in dust. (i feel like a broken record)
talent does what it can
genius does what it must
i do what i get paid to do




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