problem in my gluthatione seharose beads? - (Jun/08/2006 )

hello,
i am having problems to bind my GST tag protein to sepharose beads? purification is so bad?
i checked the pH of my wash buffer and the incubation time for binding is 30 min at room temperature(i tried 1 hour at 4 degree but still nothing )
help please

aaaanyyyyyy bodyyyy???????

how did you solubilize? sometimes strong detergents effect the binding.....another stupid question : did you wash off ethanol?

hello kathy

thankx for talking to me.
i tried yesterday adding DDT to my lysis buffer and it worked a little bit i mean i could finally see my purified protein band but still the binding effieciency may be 50 % .
for ur question yes i did wash the ethanol.
by the way i notice u are from japan, me too, well just studying.

hi again
forget to say that my protein is a soluble one.

Hi.

Could you please tell us exactly what conditions you have been using for the lysis step? Also, how do you load onto the columns?
50% binding is not all that bad. If you need to, you could always run the lysate over the column a second time.

hello swanny
i used a Bugs BUsterd lysis buffer with 1mM DDT and i didnt use the column methode i used the Batch methode ( in small tubes).
thankx

spanishflower, where do you live in Japan? i live in sapporo.....the coldest city in the world... i hope ur not finding it very difficult to study in Japan...
back to your topic, i would agree with swanny, 50% binding is not bad at all....can't you use it as it is? i would not try to optimize further....

hello kathy
thankx
i live in kumamoto.
i will try next time different concentration of DDT and i will let u all know.
thankx again

It all sounds reasonable. Are you able to elute the protein OK? (I once had great binding to a column, but then I Couldn't get the protein off again...)
If elution is OK, try running the lysate on the beads again.