hi hui-chu chang,
I'm working on a calcium transporting ATPase, which is a membrane
protein with probably 10 membrane spans. recently, we had this type of
problem which was resolved by mixing urea SDS and bMe in a special way :
15 µl of a mixture containing 6.7 % of SDS and 4.6 M of bMercaptoethanol
(in fact it corresponds to a 2:1 mix solution of 10%SDS and 14.3M bMe
(stock solution)) is mixed to 40 mg of urea (at this point, the urea is
not completly dissolved); then 40 µl of sample is added and, after 1 min
vortexing at room temperature to dissolve urea, the mix is heated for
70 sec at 100 °C. THe concentration of urea is approx. 8M.
If you want, you can see this note in Anal. Biochem. 1996 236, 363-364 .
However, it seems when I see what you describes that the problem can
come from the reducing agent that you use, I'm afraid it is too old.
pierre falson
falson at verlaine.saclay.cea.fr
