I have been trying to purify a protein (from Sf9 cell conditioned media) using anion exchange Chromatography followed by a Nickel column. After the Ni column, I buffer exchange the eluate to remove the imidazole, using a PD10 column.
However, I noticed that my protein values have dropped down to nothing after the buffer exchange to remove the imidazole. Before the buffer exchange my probes show SDS-bands and also nanodrop concentrations. However, after the PD10: nothing.
Does anyone have an idea of what could be happening. Protein precipitation? I used the PD10 in between to desalt my protein after the Ion exchange as well, and then I noticed no such issue. Its only afterwards that I notice this.
Best,
Ibrahim
You would expect some losses (up to about 20%) and significant sample dilution by doing PD10 exchange. This may mean that bands seen on gels before PD10 will be much harder to see afterwards.
The loss of absorbance is real, but almost certainly it has nothing to do with protein (other than the dilution/losses noted above). You have far less protein than you think. Imidazole absorbs at A280 and this signal 'disappears' after PD10 (strictly speaking, it moves and elutes much later than the protein).
In summary, almost certainly you are seeing reduced A280 absorbance values primarily because of the removal of imidazole, plus a further reduction because of loss of protein/dilution of protein on the PD10.
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