I am using GE healthcare poly-buffer 96 and PBE 94. I use a 0.025 M ethanolamine start buffer pH 9.4 and an elution buffer of PB 96 pH 6.0. I take care to degas all my buffers, keep the temperature constant and dialyze my protein sample into start buffer before loading and have repacked my column. I still constantly fail to get a linear elution gradient when I pass through my protein sample. Doing a blank run I have managed to achieve linear gradients. Is it possible my protein is overloading the column and causing the problem through its own amphoteric nature?
I also am confused by protocol instructions to begin the elution, then load sample in start buffer and then continue elution. Should it not make sense to load sample and then elute?
Hi Arshad
Thank you for your help.
I repacked my column and have run it again. This time with a different protein. It elutes a pH 6.5 and has done so twice. repeatedly Once with and once without the gradient again flat-lining at pH 7.8-8 for a considerable amount of time. This suggests that it was not the protein. Can it be the addition of DTT which I add to keep my protein stable though I only use 1 mM and the literature tells me is a common additive.
I should also add that though most of the protein eluted at pH 6.5 some only eluted after I began the salt wash. Both proteins are enzymatically active though. Could it be the second is partially aggregated protein?
I have washed the column with NaOH and NaCl to clean it before both runs
As always I have diluted my protein into the same start buffer I use on the column
Can i ask you, the protocol says start the elution, than load the sample in start buffer and then continue the elution. This has always struck me as strange and anti-intuitive.
Okay I think I understand in regards to elution. If the literature says that my pre gradient volume is 3 column volumes does this mean I should let the elution run for 3 column volumes before adding sample? And then won't the sample in start buffer swing the gradient backwards?
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