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how to get rid of post-translational modifications


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#1 Proteomixiao

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Posted 28 June 2012 - 10:27 AM

Hi all,

I have been working on a protein that when it is transfected into 293T cells, it has a mw of 43kDa, but the endogenous form in HSMM has a mw of 70kDa. So my colleagues and I suspect that the protein has been heaveily glycosylated. We tried to use the Protein Deglycosylation Kit from BioLabs, and we were able to bring down moderately to 70kDa species to 43kDa. Other studies show that the 70kDa is not a product after heavey phosphorylation. Now we are thinking about to get rid of all other PTMs that we can possibly remove. Can anyone suggest a viable method to try and the corresponding control that I will need?

#2 bob1

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Posted 28 June 2012 - 01:50 PM

Stupid question but, have you ruled out that this is not an error somewhere in the process, whether it be in the cloning (sequenced the clone, everything OK?), detection (same antibody with other samples gives the 70 kDa band?), expression (express in the cells with native HSMM- look for enhanced band?).

#3 Proteomixiao

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Posted 29 June 2012 - 05:46 AM

Stupid question but, have you ruled out that this is not an error somewhere in the process, whether it be in the cloning (sequenced the clone, everything OK?), detection (same antibody with other samples gives the 70 kDa band?), expression (express in the cells with native HSMM- look for enhanced band?).


Thanks for the reply.

Yes we have completely ruled out any problems in the cloning, transfection processes, and it's been established that the protein has two major species 43kDa and 70kDa ones depending on where it is expressed, i.e. 43-50kDa species only in ventricle, skin, ear (cartilage) and 70kDa in lung, liver, spleen.

#4 bob1

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Posted 29 June 2012 - 11:37 PM

Hmmm, I would suspect that either there is a dimerized form that is really strongly bound (possibly self re-folding) or there is an alternative reading frame for the 70kDa species before suggesting that there is heavy post-translational modification.

Have you looked for glycosylation sites on your protein of interest and found enough to account for the difference, given that most glycosylations are monosaccharides?

#5 Proteomixiao

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Posted 04 July 2012 - 12:00 PM

Hmmm, I would suspect that either there is a dimerized form that is really strongly bound (possibly self re-folding) or there is an alternative reading frame for the 70kDa species before suggesting that there is heavy post-translational modification.

Have you looked for glycosylation sites on your protein of interest and found enough to account for the difference, given that most glycosylations are monosaccharides?


Thank you for replying again.

We are also suspecting that it might be a dimerized form but however we have been able to bring the 70kDa down to ~60kDa with the deglycosylation kit we have currently, which makes us believe that it is more likely to be a glycosylated form. I have found 4 consensus sites for glycosylation but it doesn't account for all, plus glycosylation can also happen at regions with no consensus sequence.

However, I have not checked for alternative reading frame. But when we are able to IP the 70kDa species, we will send it for NMR analysis.




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