Protein puriification using IMAC: flowthrough very slow
Posted 15 March 2013 - 01:48 PM
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!
Posted 18 March 2013 - 05:39 AM
Posted 18 March 2013 - 07:55 AM
this principle is described in the gel filtration handbook available from ge healthcare.
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