Why DTT in RNA prep?

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Fri Sep 17 05:21:21 EST 1999

Rudi van de Wetering <nospamr.vandewetering at czzorlnm.azn.nl> wrote:
>>Maybe it is to reduce the proteins such as RNase.
>>A final concentration of 1 mM DTT at pH of 8 should be quite effective at
>>reducing all proteins.
> I read that adding DTT (0,5 mM) to cell-lysates prepared for measurement of
> B-gal stabilizes its activity. I use it routinely now and it works fine. Why
> doesn't B-gal inactivate, or Luciferase for that matter since I measure
> both? Don't they have disulfide bonds?

No; they are intracellular proteins, i.e. they occur in a reducing
environment. There are very few intracellular proteins which have
disulfide bridges. On the other hand, pancreatic RNase is a secreted
enzyme, i.e. it has to survive in an oxidizing environment. In this
environment, disulfide proteins stabilize the protein fold, and
reduction of them destabilizes the proteins (also true e.g. for


/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */

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