non-reductive gel to check the disulfide bond - (Aug/04/2006 )
QUOTE (genehunter-1 @ Aug 16 2006, 01:51 PM)
It should not be as a chemical reaction, unless it was subjected to long oxidative envirument. I assume you only briefly treated samples, right?
I didn't even use oxidative environment to trigger disulfide bond formation. Only 1xPBS incubation for 30 min at room temperature, then I saw the dimer on western blot.....Of course this dimer also appears in oxidative enviroment group, but since I can get it using PBS, why bother using oxidative agents?....Afterwards I found out that this dimer doesn't break at RT with DTT or ME, and does break when boiling without DTT...
So you suspect the dimer I got is not a proof for disulfide bond?
-bigworm-
Heating denatures protein and it break off, not due to breaking the s-s linkage. Heating is not strong enough to break it, unless under strong oxidation condition that breaks s-s.
-genehunter-1-
QUOTE (genehunter-1 @ Aug 17 2006, 01:57 PM)
Heating denatures protein and it break off, not due to breaking the s-s linkage. Heating is not strong enough to break it, unless under strong oxidation condition that breaks s-s.
I am confused, I thought reducing condition breaks s-s linkage, strong oxidation condition will also do so?
My sample loading buffer: 50 mM Tris (pH 6.8), 10% glycerol, 2% SDS, (w/ or w/o 10 mM DTT or 1% ME); The dimer disappears after boiling for 5 min w/o DTT or ME.
This loading buffer is stored at 10x concentration for 6 months at room temperature (DTT or ME was added freshly), but it should not be an oxidation condition, right?
-bigworm-
hi,
boiling is necessary, SDS no sufficient, to completly unfold the protein and in particular to expose the sites for further effective reduction of SS links...
the only dimers dissolved by non-reducing conditions are not formed by SS bond but electrostatic or hydrophobic interactions
-tryptofan-
QUOTE (tryptofan @ Aug 19 2006, 09:12 AM)
hi,
boiling is necessary, SDS no sufficient, to completly unfold the protein and in particular to expose the sites for further effective reduction of SS links...
the only dimers dissolved by non-reducing conditions are not formed by SS bond but electrostatic or hydrophobic interactions
boiling is necessary, SDS no sufficient, to completly unfold the protein and in particular to expose the sites for further effective reduction of SS links...
the only dimers dissolved by non-reducing conditions are not formed by SS bond but electrostatic or hydrophobic interactions
My situation is complicated, I am dealing with membrane protein, and it get aggregated when boiling. The problem is: I saw dimer when using room temperature and it disappears when boiling, I just don't know the disappearance of dimer is because that dimer is not due to S-S linkage or because the S-S linked dimer get aggregated more easily than monomer.
You see my problem? actually I tried 60 degree 30 mins also, the ratios of aggregate:dimer:monomer are:
Room temperature 1 hour:--------4 : 2 : 4
60 degree 30 min:------------------20: 1 : 3
boiling 5 min:------------------------ 80: 0 : 1
The dimer disappears when boiling, nomatter with DTT or not. But it may because all the dimers are aggregated, while some monomers survive.....
Any suggestion?
-bigworm-