DTT in mammilian lysis buffer? - (Sep/11/2006 )
i found a protocol talking about extraction of nuclear and cytosolic proteins in mammilian cells using DTT in the lysis buffer?
dont know why exactly?
the concentration for dtt is 0.5mM in this protocol, so if we did that this means the protein willnot be in its native form so we cant do any other binding experiments , e.g testing the ability of this protein to bind to another one???
is this right???
DTT in this concentration has protective role to keep protein from oxidation damages.
so it will not affect or denature the structure of the protein?
Probably not. Oxidation-reduction is reversible reaction and native protein is the energy favorable form. Even if it is reduced, it will go back.
ok i am sorry be patient with me
so how can i determine the concentration of DTT i mean in some lysis buffer i read 1mM and some 10 some 50mM
so do u mean that the DTT effect changed according to its concentration?
10 mM sounds little high to me. I use 0.5-1 mM most time. Too much DTT can cause trouble. Besides, it is quite expensive as well.
i found this on the net
DTT at lower concentration stabilize enzymes and other proteins which posses free sulfhydryl group and restore its activity.
so a stupied question if a protein isnot from this category (sulfhydryl) so DTT has no effect on it ? or it will denature it even at lower concentration?
this is because i asked my professor and he said DTT cause denaturation in all its concentration
I would not agree that DTT only does denaturation to protein. DTT probably does more to abnormal proteins than normal ones that are correctly folded. You are right that low concentration DTT reduces unwanted S-S bonds and keeps free SH- groups alive. Free SH is very easy to be oxidized. Other amino acid side chains can also be damaged due to oxidation. tyrosine or lysine for example.
ok my last question
how can we define ( lower concentration)
i mean does 5mM considered low or high?