Protein puriification using IMAC: flowthrough very slow - (Mar/15/2013 )
After extracting my protein in Tris-sucrose buffer, I do a dialysis to take out the sucrose. Then I centrifuge it to get rid of any precipitation. I then filter it using 45 um filters. Then add the Ni-NTA agarose to it and leave it overnight at 4 degree celcius. When I run it through the colum it goes very very slowly like 1ml in 1 hour.
So it basically takes me like ages to purify my protein.
Can anyone please suggest to me what I can do speed up the process? Other than using pumps as my lab don't have one!
Based on how you prepared your sample, I would say you have a problem with your column. Maybe your frit is clogged or is too small of a pore size or your drain port is clogged. Your flow rate is way too slow even for gravity flow and your sample shouldn't be causing that issue given the way you've prepared it.
since you appear to be using gravity flow, you can make the exit tubing longer and set the receiving vessel lower to increase the flow rate.
this principle is described in the gel filtration handbook available from ge healthcare.