Ghost band in SDS-PAGE - Help me!!!! (Mar/20/2006 )

how can you be so sure that your protein is pure?
how did you purify?

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I'm so sure cose I performed an Edman degratation analysis on the protein eluted from that band!
And it had the same AA composition of my protein!

Today I read something about these problems on internet and now I think it could be the result of a "non- covalent aggregation, sds-resistant". What still I don't know is how to prove it!
I mean: if it is not covalent but so strong to resist to the action of SDS, what could destroy it?


Any idea?!?!?!?

Thanks again!!!!!

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I had similar problem with one of the RGS. Finally it turned to be heavily glycosilated form of the protein. It is easy to prove: just transfer onto membrane and make Western.
Good luck!

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Maybe I didn't understand what do you mean but if is a sds-resistant non covalent bound between the subunits of my protein, what can I prove with a western blotting? At the end is still a "strange form" of the same protein and I don't know if the western can recognize this difference!

I'm sorry I'm not an expert of this technique

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you can analyse glycosylation of your protein with some kits like this one.

http://probes.invitrogen.com/servlets/publications?id=225

you could also treat your cells (if it's from cell culture) with some enzymes that cut glycosylted groups . Sorry, I have no example in mind right now

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What Taras Bulba want to say is that your ghost band may be caused by glucidic chains attached to your protein. Is it capable of being glicosilated? you can check this by analising its pimary aminoacid sequence.

For example, Asn-X-Thr/Ser will be N-glycosilated if produced in mammalian system. So, if your protein has glucidic chains, it may co-purify with the unglycosilated isoform, and the MW will increase.

But a question come to me: if all molecules have the same primary sequence capable of being glycosylated, all them will present glucidic chains, will not them?

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I mean that probably both bands on your gel are the same protein but one of it - before and the other - after posttranslational modification. In my case it was glycosylation but can be something else. By Western you can prove that both bands are the same protein . If so, your next step is to analyse the modification. If not - it can be potential binding partner.

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Quick question, do you treat the DTT itself before using it? Try treating the DTT with tributylphosphine. This reduces any oxidised DTT. The make up your loding dye to 1.4% DTT and 7% iodoacetamide. Both these treatments are generally necessary to get the 'most' out your DTT

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Yes I think so....if they are glycosylated, both of them should be glycosylated!!!!!
So maybe is not this the problem!!!!!

But I will check the primary sequence!!!!!!

Thanks!!!!!!!!!!!!!!!!!

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Thank you very much, this is very interesting!!!!!!!

Do you think treatment with tributylphosphine is always necessary? Even if the DTT is just bought?!?!?!

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