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

hi again
swanny i amnot doing elution for my protein as i am using it in another way.
i still use GST protein for further pulldown assy.
still i add 10 mM DDT and make the incubation time 2 hours at 4 degree with slow rotation but still the binding capacity is not that good??????
any ideas please__

hi

i have found that column purification is more efficient than beads

then again u can try what some one eariler suggested
recirculate the lysate
try increasing the concentration of elution buffer , keep ph steady between 8-9

i have attached some amersham handbooks on the topic in another topic on GST inthe forum
download and see if they can help you

alll the best


regards
laxmi

Hi:
you may incubated at 4 degree overnight and then wash, elute

hi guys
thankx
i have a question , have anybody tried BugsBUsterd lysis buffer?
what kind of lysis buffer do u use?

Sorry, we have not used bugbuster lysis buffer for the high price, we use sonication.

thankx for ur reply

guys .... i amnot doing elution of my protein....please help
after the binging with sepharose beads i wash 3 times with PBS and then take an aliqute and proceed to western blot ( i add lameali sample buffer and DDT to my sample then boil) no baands are there
my all GST protein doesnt bind to the beads......