Getting rid of background protein binding on GST beads - (Mar/22/2009 )

HI everybody,


I Have a problem with getting rid of the background when i use glutathione sepharose beads to bind my GST protein. Should I add EtBr to my wash buffer? I have read in a few articles that this discourages protein from binding DNA. I was thinking that DNA could be the cause of teh non-specific binding. I am also thinking should I block with 5% milk before incubation with my GST protein. How do I determine the blocking buffer composition and for how long and at what temperatures are suitable?

Just wanna take the opportunity to say thanks for all your help. This forum is too good to be true.

Hey do this:

Wash extensively with wash buffer which has 500 mM NaCl. If its non specific binding, it should go.

Best,
TC

T C on Mar 22 2009, 08:48 PM said:

Hey,

I think the non specific binding to the beads is due to ionic interaction of non specific proteins with the beads. When you add NaCl, the strong ions compete with the non specific proteins and dislodge them from the beads. However, I am not sure about the theory, I accidently discovered that NaCl works and later found many people doing it. Target protein is not affecting as its affinity purification.

I use high NaCl in the wash buffer and not in the elutions. AT at the wash stage, the high salt doesn't affect most of the proteins but it will entirely depend upon your protein of interest.

I am not sure about any text which covers protein-protein interactions alone but being a chemist I always start with voet, stryer, lehninger and all basic books as they are easy to understand.

Best
TC

I still get the background reading. There seems to be no difference if I was with PBS, PBS with extra NaCl ( up to 500mM). Perhaps my reaction volume is too small. I use 40ul of beads slurry and 200ul of lysate.

I also wash 3 times as per recommended in the GE healthcare booklet for the glutathione sepharose 4B beads. Should I increase the number of washes?

Perhaps I should increase the amount of salt in my lysate. I.E. the buffer I use to lyse the cells since the non-specific binding could be due to ionic binding.

Has anyone tried adding EtBr to the wash buffer. Perhaps there is alot of DNA in my lysate. Thus the high non-specific binding. i.e. more DNA binding to beads, protein inturn binding to the DNA?

Should I also try to wash the beads with PBS + high salt before binding? to get rid of ionic interactions?

Hey,
Dilute out the lysate to say 1 ml or 2 ml and bind and check. I use 150 mM NaCl in the buffer used for lysis. Then wash extensively with buffer containing 500 mM NaCl. Dilution won't won't really affect the binding of target protein as its affinity purification. If you want to precipitate the DNA, you can use 0.1% PEI or DNAse.

ANother option is to go for another step of purification and do FPLC after you purify by affinity purification.
Best,
TC

MaggieRoara on Mar 25 2009, 08:00 AM said:

I still get the background reading. There seems to be no difference if I was with PBS, PBS with extra NaCl ( up to 500mM). Perhaps my reaction volume is too small. I use 40ul of beads slurry and 200ul of lysate.

I also wash 3 times as per recommended in the GE healthcare booklet for the glutathione sepharose 4B beads. Should I increase the number of washes?

Perhaps I should increase the amount of salt in my lysate. I.E. the buffer I use to lyse the cells since the non-specific binding could be due to ionic binding.

Has anyone tried adding EtBr to the wash buffer. Perhaps there is alot of DNA in my lysate. Thus the high non-specific binding. i.e. more DNA binding to beads, protein inturn binding to the DNA?

Should I also try to wash the beads with PBS + high salt before binding? to get rid of ionic interactions?