We wish to separate unconjugated peptide after coupling peptide and thiol using Sepharose CL 4B suspension. As we have Sepharose CL 4B suspension, kindly advice how to pack a column using the same.
Thanks in advance
Ur question is very general. Gel selection mainly depends on the molecular weight of desired product and starting peptides. Similarly your mobile phase depends on the solubility of ur starting material preferably aqueus buffer. Sepharose is itself is stationary phase, no logic of mixing it with silica. If u provide more details, i may guide you in better way...
Hi Preeti,
Agree with Ganesh above. We need more information on your experiment question. In general, unreacted peptides will remain in the supernatant, unbound. If you are trying to characterize the unconjugated peptides, you can use a Zip-Tip kind of manual, reverse-phase chromatography to purify and analyze them.
Hediye
Packing a sepharose column is indeed tricky. There used to be nice booklets from Pharmacia, but I have not seen them for years.
The trick is you must equilibrate the sepharose in the buffer you'll use for separation, then you need to eliminate the fines, then pour without formation of preferential pathways.
So
- measure the column volume, take 1.5 time that volume of Sepharose, carefully resuspended in its superrnatant (I think it is still sold wet);
- put it in a void flask (kitasako flask), leave to sediment, carefully remove the liquid, replace it by a large volume of your buffer, mix, leave to decant, carefully remove the supernatant liquid, and repeat at least twice. We used to do this using a glass pasteur pipette collected to a water pump and care; this also eliminates the fines as they sediment slower than the rest;
- now connect the water pump to the kitasako to degas the Sepharose phase
- prepare your column with a funnel on top (lab suppliers sell funnels adapted to their low pressure chromatography columns), check that it is vertical, fill with buffer up to about 1/2 or 1/3, close the exit.
- resuspend the Sepharose in the buffer to get a thickish and homogeneous suspension (taking care not to incorporate air) and pour slowly but without stopping until all the gel is in the column/funnel combination
- apply pressure (a bit higher than your operating pressure) and let run for a night
- remove funnel, close without leaving any void volume, run a verification run with dextran blue (if there are preferential pathways, this will highlight them).
Good luck!