I have been trying to purify recombinant p300 Histone acetyltransferase domain (expressed in E. coli BL21 strain )using Ni-NTA His Bind Resin. At first I used 37 degree C temp 3 hrs for IPTG induction. However, while troubleshooting I changed it to 30deg C for 4 hrs (since lower temp works well for enzyme). Obtained good induced bands in both the temperatures. All the buffers used for the experiment are standardized in our lab. Sonicating the bacterial pellet at 32% amp, 3sec ON 5sec OFF, 4-5 cycles (4 cycles when culture volume was 100ml and 5 cycles with 500 ml volume culture) of 1 min sonication with 5 min gap. Used 500ul of Ni-NTA bead slurry calculated according to the technical datasheet. I have observed p300 HAT domain bands (68 KDa) in the flowthrough and wash 1 fraction but bands just below 50 KDa marker were present in all the three elutes and beads. Before checking the activity of the elute fractions, I want to know the possible reasons for such things.

Hoping to get responses.

Thanks in advance.