Probelm with gel filtration - (Aug/10/2010 )

Hi all

I've faced a problem with gel filtration.

I use Superdex -75 colunm, the buffer is 50mM Tris pH7.25, 150 mM NaCl.
the protein is fused to GST. It cut nice from GST using PreScission. the problem is the GST and protein of my interest "11 kDa" appeared in the same fractions. in this case does the salt concentration could affect the purification because I used 300 mM NaCl before and they appeared separately.
The first run that I did for this protein seeded very strange where my protein 11 kDa came out the colunm before GST 26kDa (50mM Tris pH7.5, 150 mM NaCl).

Thanks in advance

Your post is a little confusing...but....

G-75 is not good enough to separate the fragments that you are looking for as they are close in molecular weight. You are much better off by getting some glutathione beads and binding your free GST leaving your cleaved protein in the supernatant...probably a lot faster and easier method....

Thanks for your reply.
What about PreScission protease. Do I need to apply the sample on chromtography like gel filt or Ion exchange to get rid of other protein especially PreScission.

Thanks

I think your results of the first expt is correct because smaller ones elute faster than the larger ones. I guess it happens with the GST tags, cos I too faced the same problem. I tried binding the GST to the resin, but din't work. I had to simply change the tag! Poor me:( You can try to use a different column instead, since the MW difference is less.

shivasankari on Thu Nov 18 15:01:18 2010 said:

you are thinking of electrophoresis. with gel filtration the larger protein comes out first.

if your 11 kDa protein is coeluting with your 26 kDa protein then you may be overloading the column. you can try running less.

you can also try superdex peptide. the 26 kDa protein should come in the void and the 11 kDa protein should fractionate.

the amount of salt may make a difference depending on the protein. it's possible that the 11 kDa protein interacted with the matrix at the lower salt concentration while the 26 kDa protein didn't.