blurry bands on SDS-PAGE
peter.cherepanov at uz.kuleuven.ac.be
Fri Mar 8 05:22:30 EST 2002
may be these are obvious:
1) I can bet on salt: sodium sulfate or ammonium sulfate in my samples
(after HIC) give me horrible smeared bands, TCA precipitation takes just
half hour to make the gels look just perfect.
2) There are examples of proteins normally stabilized by disulfides and
membrane proteins that behave strange on SDS-PAGE gels.
One example - HBsAg, it needs to boiled for 30 min with lots of DTT+bME, and
even then it is not completely reduced.
Other example - HBcAg, it may not be boiled at all, or you just see a weak
smeared band and all the rest stays up; but it must be reduced before
loading onto a gel, or you do not see a band too... (reducing at 37oC is
good in such cases, some membrane proteins, I heard, behave in a similar
way). Some people say that it is helpful to add bME into the running buffer.
I think, there is always an excess of APS (persulfate) left in the gel, and
since discontinous system can not be pre-run, it might give a problem with
easily oxydizing poplypeptides.
3) pH - if your protein is Cys-rich, and you use unbuffered TCEP to reduce
it, then pH of your sample will get so low that buffering capacity of the
gel will be not enough. In fact, adding TCEP is same as adding an equal
molar amount of sulfuric acid to the sample. You can judge by the volume of
Tris you will need to turn your sample from yellow to normal blue (like our
4) may be indeed heterogeneity in structure ... (are proteins really
completely denatured in SDS? I bet not, some protein have been renatured
properly in sarkosyl or SDS with correct disulfides formed).
5) glycosylation may will make your band not sharp or smeared.
I have feeling you know most of it yourself ...
Good luck anyway,
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