The membrane glycoprotein which is made up of 79% lipids and 20% protein whenever we go for SDS PAGE with fresh sample will give clear profile in some samples and not with other samples. Simultaneously storing 1-2 days at -80 or 4 causes fat ball formation and not coming out of stacking gel well. The delipidation will affect the protein concentration and not giving proper SDS PAGE profile. we we tried with 10%, 12% 15% gel same thing. any body can suggest how we can get good SDS-PAGE profile for membrane glycoprotein other than normal trouble shooting of SDS PAGE
Thanking you
The role of SDS is to denature a protein into as tight a ball or spheroid possible so they are all approximately the same shape and importantly swamp the surface so they all have the same charge to mass ratio. Thus when the electrical current is applied they all move at the same speed (i.e same mass to charge ratio) and are resolved according to their overall size which should be proportional to mass as they all have the same shape (e.g. are either big balls or little balls of proteins). The trouble is that the sugar side chains are huge and don't interct with SDS in the same way as amino-acids in a peptide chain. As a result glycoproteins are not perfectly rolled up into tight balls, and the terminal sugars are sometimes charged and thus alter the perfect matching of charge to match ratio fundemental to SDS-PAGE electrophoresis.
Article Comparative studies on the analysis of glycoproteins and lip...
Hi Guys !
The majority of issues related to glyco protein in SDS-PAGE is mainly due to heterogeneity in glycosylation. By the way, not all glycoproteins produce a smear on SDS-PAGE. If glycosylation is relatively consistent (as in the case of RNAse B, to cite one example) the protein will produce a single band on SDS-PAGE.
Heterogeneity of CHOs is not random and meaningless, and It might be worth digging in to, despite the disorder, interference with rational thought, and lack of aesthetics. In almost all cases we do not know what CHOs are really doing and beyond that we do not know what the "use" of heterogeneity is--but in fact it many help answer the first uncertainty.
Another rambling thought: carbohydrate molecules attached to a polypeptide broaden the range of folded forms possible for the polymer (however slight the differences), and/or expand the degree, nature, and range of interactions of the polymer with other molecules--polymers, drugs, metabolites, other cellular components, gel and buffer molecules etc. But what are the forces at work? If Tricine eliminates variation, how and why is it different from a conventional buffering agent? Is the variation and its elimination purely an experimental artifact of no biological significance ? ..to be continued.
An important paper about protein glycosylation in SDS Electrophoresis Process is attached !
Good luck
Zoheir
Lipoproteins will be very difficult to resolve by SDS PAGE as it's charge based separation. I suggest use of a gel permeation column instead. If size of this lipoprotien is less than 100 kDa use Sephadex G-100 to separate lipoprotien on the basis of it's size i.e. molecular mass.
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