problem solved

redeamer aniko at ut.ee
Sat Jan 10 08:51:15 EST 2004


Hi and thanx for help!

I have attempted two strategies (both yielded a denatured probes):

1)"titrated" out K+ with SDS, adding SDS to my extracts and
centrifuging down all the whitish stuff, my samples were ok afterwards
on the gel and i got some of my proteins on western blot.

2)TCA precipitated them (final 10%), incubated on ice 1h, then
pelleted, then acetone wash step (ice cold incubated 15 min on ice,
pelleted), however all the TCA have nt been washed out and the pellet
almost refused to dissolve, however, i could monitor the solvation by
change of the pH of Bromphenol and titrated in back with 1M TrisCl
pH6.8. Well, i had a lot of unpleasant experience with dissolving
protein pellets, but i have now 10 proteins of 17:-))

So everything worked, but the easiest thing to do was the first one:)
But i was so devastated after 2 overnights that it didnt come to my
mind:-)

Thank You for suggestions,

Drew



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