Protein Query

P.C. PC at nospam.edu
Sun Mar 14 13:17:13 EST 2004


what are the concentrations you are talking about? Can you guess from 
the activity ? Do you know the sequence?

can you detect it (in your peak fractions) by UV @280nm, Bradford or BCA?


Peter




gilbertjack at hotmail.com wrote:
> This is an update.
> 
> 
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> I am currently working on a bacterial protein which does not seem to migrate through an SDS-PAGE or Native-PAGE at all. In SDS-PAGE it get stuck at the stacking/resolving gel interface. in Native-PAGE it hardly leaves in the Stakcing gel.
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> It will only stain with Stains-all, Coomassie and Silver staining techniques (and most others i have tried) have no effect.
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> We can track this protein by activity, and activity is lost following treatment with proteases (pro K and Pronase, but not Trypsin). therefore I am pretty sure it is a protein.
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> The non-migrating band on the gel increases in intensity with increased specific activity following multiple purification steps designed explicitly for the purification of the protein. It is the only band which increases in intensity on the gel with increased specific activity, hence, I am somewhat sure that it is our protein.
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> One more piece of info, the protein stains pink with stains all on an SDS-PAGE following boiling, but blue if you run a native gel. The protein is also calcium dependant.
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> We have tried looking at it via MS Maldi-QTof, but we see nothing of interest. We can digest it with Trypsin if followed by excessive detergent treatment, and MS of this shows only one obvious species at 3.5kDa.
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> Honestly I am reaching the end of my teather with this protein. If anyone has any suggestions as to why it may not migrate in a ployacrylamide gel or any suggestions on how to sequence this protein I would greatly appreciate it.
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> http://biowww.net/mynews/tree.php?group_name=bionet_molbio_proteins&begin=0




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