Superdex or Sephacryl? - protein purification by size exclusion chromatography (Oct/25/2007 )
Hi,
after His6 purification (Ni-IMAC resin) I'd like to separate my protein fraction from smaller proteins using a size exclusion chromatography.
My protein has a size of ~72 kDa, contaminating proteins are still present at ~50 kDa, 40 kDa, 30 kDa but in a very low concentration. At this point I am not sure whether I should use Sephacryl media (i.e. S-100 HR column) or Superdex (i.e. S75). When I tried to pack a Superdex 200 column by myself the separation did not work in principle.
Can you recommend me one of it?
As I have to order a new one there is no chance for a tryout in advance.
Thank you for your help!
gel filtration for proteins with this close difference (70 kd vs 50 kd) is possible. You may have some overlap between the two, and the procedure is sample volume limited (less than 5% column volume)
Superdex has higher resolution, but is more expensive than S-100.
Sometimes, an ion exchange column can be very efficient and is faster, and less dilution for your sample.
hey!
thanks for answering. I tried several things from that point:
- purification using Sephadex 200 column
- purification using Sephacryl S-100 column
In both attempts I could not separate my proteins. Although I get a very nice spectra the target protein smeers over almost all fractions. My impression is, that the proteins stick somehow together. At the moment I use standard conditions: 150 mM NaCl, 30 mM Tris-HCl pH 7,6.
What do you think, shall I increase the salt concentration to reduce possible ionic interactions between the proteins? May it be possible to add a detergent to increase the solubility of proteins?
Has anybody an idea what could happen here? First I thought the S200 column is somehow defect, but I got the same result using the S100 column.......
Did you get good separation with the MW standards? Sounds like you either have protein solubility problem, or some ionic interaction. What is the pI of this protein? Raising [NaCl] to 0.5 M see if that helps.
my protein has a pI of around 6.5. You're right, an IEX column would be a good try out. For Gel filtration I also thought about a salt conc. of 500 mM. Did you ever work with detergents during gel filtration? I did only once following a protocol......
For protein with a pI= 6.5, non-specific ionic interaction should not be a problem.
Again, did you get a good separation with standards?
Separating these two proteins can be tricky, though possible. You need to have a very good column and you need to keep sample volume low, like 2% of the column volume. You normally need to check the column by runing through blue dextran 2000 to see if it is well packed. You may also want to check with MW standards, individually run for each protein.
hi there,
I've tested the column using blue dextran and two other MW standards in my buffer conditions. The loaded volume is below 2% of the column volume. Although I've packed one of the column by myself, it worked very well for the standards. I'm a bit puzzled, that its not working.