Centrifugal Filter Unit - Trying to get a higher concentration. (May/17/2010 )

More and more and more problems...

My protein is 37kDa and the ultimate plan is to make an antibody against it...
I have purified enough protein, but the concentration is way too low (about 180ug/ml).
I used centrifugal 30 MWCO filters at first to get the concentration higher, my protein leaked out.
Now I switched into 10 MWCO filters (Amicon Ultra-4 Centrifugal Filter Device)... it seemed to be working better at first... now I have about 0.5ml sample 760ug/ml... which is way too low... and then I still have about 30ml sample of this 180ug/ml stuff...
I have tried to use the filters, but the lovely protein is leaking out...
silver staining and western blotting shows the right 37kDa band, so according all the protocols and manuals, this should be next to impossible.

This centrifugal filter has no troubleshooting... so any suggestions what might be the problem this time?

I WOULD SUGGEST USE A PALL 10 KD CUT OFF FILTER.. U CAN USE A 15 ML ONE SO THAT THE CONCENTRATION IS FASTER...
ooh sorry abt the caps!!!

also you can lyophilize it and resuspend in the required volume if that is not a problem!!!

Maybe try a series of MWCO filters, rather than trying to do it in one step?

especially when trying to pass a large volume through a small filter you will get protein sticking to the filter. flush the membrane with the sample as you remove it so that you resuspend any protein stuck on the filter. you may also want to concentrate the solution in smaller aliquots to reduce the amount of protein that sticks to the membrane.

also, with what is your protein in solution? you need to maintain a minimum ionic strength of 0.15-0.2M (depending on the type of membrane) to reduce non-specific binding to the membrane.

30 kDa cutoff is too close to the size of your protein. 10 kDa cutoff is the right porosity (assuming that your protein is globular).