Hi there!
I'm purifying a recombinant protein and I always get 2 spots in SDS-PAGE in which one spot has two times more mass than the other. Probably it's a dimer.
I would like to try an oxidative sample buffer so I would be able to break possible disulfide bonds between the protein chains.
Do you know a protocol for preparing an oxidative sample buffer?
Thanks!
β-Mercaptoethanol (5%)
Bromophenol blue (0.02%)
Glycerol (30%)
cautionSDS (Sodium dodecyl sulfate) (10%)
cautionTris-Cl (250 mM, pH 6.8)
So instead of DTT β-Mercaptoethanol
Good luck
Add 2 to 5 mM DTT , and after 15 min add 2 to 3 fold excess amount of iodoacetamide and then load your sample. Most likely, this will solve your problem.
β-Mercaptoethanol (5%)
Bromophenol blue (0.02%)
Glycerol (30%)
cautionSDS (Sodium dodecyl sulfate) (10%)
cautionTris-Cl (250 mM, pH 6.8)
So instead of DTT β-Mercaptoethanol
Good luck
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