I am trying to purify and refold my his-tagged protein on ni-nta column using urea gradient from 8M to 2M at room temperature. The buffer also has 0.25% Triton and 5mM BME. Then I wash with a buffer without urea, Triton, and BME and then elute with low pH (4.0). I am using gravity column, so it's quite slow and takes me several hours. I am consistently getting a very low yield (only around 1%). I checked the flow-through and the column for protein by SDS-PAGE, didn't see any bands. I think the protein might be degraded on the column, however, it is supposed to be pretty stable even at room temperature. Does anyone have any suggestions on what I could try to get better yield? Perhaps, I run the column for too long? I know that EDTA and PMSF inhibit proteases but I think they will also react with the column, so I can't use those. Did anyone try using, for example, pepstain or benzamidine with ni-nta?
Please help figure out the problem! Thanks.