Precipitate formation of BSA + DTT - (Jul/14/2006 )

Hi everyone,

I would like to ask how precipitate forms while mixing BSA with DTT but not with 2-mercaptoethanol?

Thanks a lot in advance.

Alys

hi,
can u be little more elobarate?
like wat conc of DTT, wat is the BSA quantity
same for beta-ME and wat r the volumes?

thanks
payeli.

Hi,

The concentration of DTT used is 10mM
The quantity of BSA used is 1mg/ml
and the amount of 2-mercaptoethanol added is 70mM.

Thanks.

Could you describe the appearance of the ppt for us? Is it gelatinous-like or something else? Can you estimate if it was most of the protein precipitated or only a small fraction? Any sign of extreme pH as a result of DTT addition?

Hi,

The precipitate formed is white in color and in the shape of flakes. I'm not so sure about the amount of protein precipitated from the solution as well as the pH of the solution upon addition of DTT.

Thanks.

Could it be that you have urea present in high conc in the sol'n that you did not notice? Did you see this when it was colded on ice or something? Is the ppt reversible when warmed up? That ppt doesn't sound like proteineous to me, at least not at this low conc (1mg/ml). DTT would not be a problem either. I am guessing it is inorganic or urea. but not sure about it. What were the other components in that mixture? Anything in high conc? humm, more info could help here.

Nope, there isn't urea present in the solution. The precipitate forms in a few hours either in room and cold temperature. The precipitate would not redissolve as well.

The only other compounds inside the solution are in low concetration (around 20mM) like Tris-HCl and EDTA.

Could this be possible: DTT broke the disulfide bonds of BSA then reoxidation occured --> led to oligomer of BSA? Can you run a native PAGE gel see if that was the case?

hi,
i have 1mg/ml BSA solution in PBS. and added 10ul of 1M DTT. i cud see small thread like structures after careful observation. u meant this kind of precipitation or different one.

if u donot mean minute thread like strucutures, try to use fresh DTT and BSA solutions and see wat happens!!

gud luk
payeli

hi,
i can think of DTT reduction reaction on disulfide bonds. but i donot think this reaction will lead to oligomerization of BSA because, to break peptide bond a system need lot of energy.

regards
payeli