2D smear problem->maybe glycoproteins

[Bradley Turner]

Hi Sebastian,

Just another suggestion, it could be a glycoprotein (or a   
protein rich in Ser, Thr or Pro).  The glycoproteins
that I study (mucins and some of their peptide fragments) behave 
similarly. i.e. on an IEF gel they will produce a smear from the cathode
to the anode.  For some reason they don't focus well, maybe because of 
the glycosylation, polydispersity, or amino acid composition (high in
Ser, Thr and Pro which I suspect may not contribute to as well a defined
pI as other amino acids).  This happens even when these peptides are
expressed in e. coli (where there is presumably no glycosylation,
hence my specualtion as to the effect of amino acid composition)

Also, these glycoproteins  and peptides do not stain at all
(or at most very poorly) with silver or coomassie in gels or gold,
india ink, amido black, coomassie, ponceau on blots.  (again
possibly due to their skewed amino acid composition) They can be 
detected very well with specific lectins, antibodies or PAS (periodic
acid Schiff) based staining.  (there are silver and fluorescence based
PAS stains that detect glycoproteins well also)

I have also found that some of the new fluorescent general protein stains
from Molecular probes (SYPRO RED AND SYPRO ORANGE) stain some of the glycoproteins 
and the non-glycosylated peptides along with other proteins.

If you are interested further, let me know and I could share some
references or methods with you.

I hope this helps,
Brad Turner


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		    Bradley Turner
	        Beth Israel Deaconess
                    Medical Center

Harvard Medical School		617-667-1215 phone
Division of Gastroenterology	617-667-2767 fax
Room Dana 536			bsturner at mbcrr.harvard.edu
330 Brookline Avenue		bturner at bidmc.harvard.edu
Boston, MA 02215, USA		turner at sprcore.bidmc.harvard.edu
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