Hi there. I have been trying to concentrate my protein with an amicon millipore ultra-15 (3 kd) centrifuge unit and my protein keeps seeping through. The buffer I am using is 6 M guanidine, 100 mM citrate at pH 3.8. I cannot decide if it's the denaturant or the low pH that is disrupting the concentrating. Any thoughts???
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Posted 20 June 2011 - 09:57 PM
dustbunny000, on 20 June 2011 - 12:59 PM, said:
Hi there. I have been trying to concentrate my protein with an amicon millipore ultra-15 (3 kd) centrifuge unit and my protein keeps seeping through. The buffer I am using is 6 M guanidine, 100 mM citrate at pH 3.8. I cannot decide if it's the denaturant or the low pH that is disrupting the concentrating. Any thoughts???
Hola, looking the chemical compatibilitis of this systems they are resistant separatelly at HClguanidine and mildly concentrates acids and alcalis. the problem to me is the density of solution and you would have the same problems at pH7. This is observed in under pressure ultrafiltration devices of amicon too where the flow trougth the membrane of this solutions is slower than aquous solution. Look in your institution for a little (there is one of 5ml) amicon stirred ultrfiltration cell and check the difference, probasbly with some effort you could filter your sample. If would be better if you made ultrafiltration after refolding if you want to do it. Buena suerte
