Is DTT better then B-mercapto - (Aug/16/2006 )
Is DTT better then B-mercaptoethanol for sample preparation fro SDS-PAGE????
and esp if the protein is highly hydrophobic and glycosylated and membrane bound???
I don't boil sample after adding lammeli's buffer so how long should I keep it (at RT) before refrigerating???
thanks.
As good as I know there's no (or very little) difference between DTT and b-mercaptoethanol.
People choose DTT because it don't smell that bad. 
I've heard that DTT is more stable than beta-mercaptoethanol.
why don't you boil your sample? I'm not sure it's efficient.
why don't you boil your sample? I'm not sure it's efficient.
I cannot boil... it is very much prone to aggregation... when I run gel... i generally get band at mol wt approx 37-40kDa(desired weight) (where i get a faint band) and a prominent band at approx 75-80kDa (which is always there even in transfected cell but not in non transfected cells)... same happens in native cell.
DTT is supposed to be stabler and i heard somewhere, stronger than mercapto ethanol.
DTT only reduces at pH near to 8. Shifting it pH strongly disminish as reductor. i.e. TCEP is used as reductor at midly acidic pH. DTT has to be prepared fresh or freezed because it reoxidize rapdly with the air.
DTT is definately more stable. UV light can degrade B-mecaptho very quickly if left in an unprotected bottle or vial.
DTT is a weaker reducing agent than 2-ME, but significantly less smelly. If you can't boil, try heating to 70 degrees C for 5 min - works well for extremely heat labile CFTR.
DO NOT keep your proteins at room temp or 4 deg C, they will degrade quite quickly, even in reducing buffer. Aliquot and freeze is the best option.
DTT has a shorter half-life than BME but has a low volatility. Both have a big dependence of pH. At pH 6.5 (20ºC) BME has a half-life greater than 100 hours and DTT 40 hours. At pH 8.5 (20ºC) half-lives reduce to 4 h and 1.4 h respectively (Stevens et al., 1983).