lower molecular weight aggregates and shift in pI

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vvg 01
vvg 01's picture
lower molecular weight aggregates and shift in pI

Hi,

When I run my protein which has 3 consensus sites for N linked glycosylation on a IEF gel, I see multiple bands, very close to each other. What do these bands mean?

Also, when I run the lower molecular weight aggregates of the same protein the multiple bands decrease in pI in a regular manner. These could be glycosylation isoforms of the same protein. Can someone explain the shift in pI?

Thanks in advance,
vvg

Chin Fen Teo
Chin Fen Teo's picture
 Hi vvg01,

 Hi vvg01,

As you mentioned that your protein of interested contains 3 consensus sites, the multiple bands your observed suggest several things which all lead to the microheterogeneity issue that is frequently found in the beauty (or nightmare, depends on which camp you're in) of glycobiology:
(1) The occupancy issue-  You can get protein with non, 1, 2, or 3 glycoforms.
(2) The complexity in term of the glycan found- For N-glycan, you can have high-mannose, complex or hybrid structures, and for the last two, you can have various extension...
(3) Remember there is no really a consensus site for most of the O-glycosylation, thus, aside for the N-glycosylation, your protein of interested may undergo one or more type of O-glycosylation...

Whereas for the shift of pI, certain carbohydrates also carry charge (in mammalian N-glycan, sialic acid will be the most likely candidate with a negative charge) that can lead to such phenomenon.

Sorry for this belated reply and hope it helps. Good luck.