Cannot get rid of GST tag - (Oct/19/2007 )
I express a GST fusion protein in pGEX vector.
I get a lot of soluble fusion protein in the elution and sucessfully cut it with prescission protease from GE.
However, after extensive desalting and dialysis, I pass through the mixture of free GST and the digested protein to the re-equilibrated GST column. A lot of GST stick to the column but there are still a lot of free GST left in the flow through.
I also try to pass through the mixture to a Q column but the pI of my protein is close the GST and they elute together even in long gradient.
The GST protein which do not bind to column probably due to free glutatione occupying the active site. Since protease is expensive so I don't want to do on-column digestion in my 25ml column.
How can I get rid of the free GST? I want to see single band in SDS-AGE.
Thank you
anybody help?
how close are the pIs? can you adjust the pH of your Q column to between them (then one will bind and the other won't)?
or
you could adjust the pH further away and see if you can elute them separately.
or
instead of a long gradient try a shallow gradient (eg .1-.15M).
I get a lot of soluble fusion protein in the elution and sucessfully cut it with prescission protease from GE.
However, after extensive desalting and dialysis, I pass through the mixture of free GST and the digested protein to the re-equilibrated GST column. A lot of GST stick to the column but there are still a lot of free GST left in the flow through.
I also try to pass through the mixture to a Q column but the pI of my protein is close the GST and they elute together even in long gradient.
The GST protein which do not bind to column probably due to free glutatione occupying the active site. Since protease is expensive so I don't want to do on-column digestion in my 25ml column.
How can I get rid of the free GST? I want to see single band in SDS-AGE.
Thank you
How much of the GSH-Bead material do you have?
Would it be possible to add an excess of GSH-sepharose to the mixture of GST and the cleaved protein in a batch mode, and later just spin down the GSH-sepharose? By this you would not change the volume of you "elution" fraction and you could use more GSH-sepharose to increase its binding capacity for free GST. The used sepharose can later easily be regenerated and re-uesed. And also be this you don´t have to care about pI values.. ;-)
The GST beads are already in excess
I can't even separate GST and the cut protein with a MonoQ column... weird..
After dialysis, free glutathione supposed to go away, and GST can stick to GST beads, why GST doesn't bind?
it's driving me crazy.
Since the system is designed to employ PreScission protease while in the column, your results will likely not improve using the proprietary materials.
You could simplify things and run a preparatory SDS-PAGE. Then simply extract the desired band.
I would not run preparative SDS-PAGE because SDS denatures my protein
Im following the protocol according to GE's GST protein purification handbook.
They describe two approaches, one for on-column cleavage and the other one is clevaged of eluted protein(such as prescission protease, thrombin, factor Xa). It said after dialysis or desalt and pass back to the GST column the GST suppose to go away(and prescission protease can stick to the column).
I also tried to pass to pre-packed Hitrap GST as well, but GST still not stick to it. The same thing happens to my labmates as well.
Anybody has similar experience that the GST tag is only paritial removed after passing the GST beads? I think this is a common problem because many people use pGEX vector too.
You could simplify things and run a preparatory SDS-PAGE. Then simply extract the desired band.