Hi all,
I have a problem with the high pressure of my HisPrep column. So these are the steps did before I loaded the sample:
1. Frozen cell pellet was resolubilized directly with lysis buffer containing 8 M urea
2. The cell lysis was done using High Pressure Homogenizer (4 cycles)
3. Sample were centrifuged 7800 rpm, 30 minutes, 4 degree Celsius
4. Supernatant filtered using funnel + paper filter (0,22 um pore size)
Reason of not using syringe filter: sample clogged very easily after even 50 mL, and it was like so hard the sample did not even go through (and I have 400 mL lysate). I haven't tried using a vacuum filter as I am afraid there might be urea residues that go into the pump - though I am not sure of this
5. Sample was loaded on 20 mL HisPrep column (as the target protein has His tag)
NOTE: As my protein is in the inclusion bodies, I also tried osmotic shock for cell lysis and did many washing steps with Triton X-100 and 1 M NaCl before resolublization with 8 M urea. The cell lysate was a bit more clarified but at some point during loading the pressure also increased a lot. Therefore I assume that this is because of the lysate that contains urea.
However the problem happened during loading where pressure increased very often and I had to reverse the column every now and then (as it was pushing the column matrix). I have observed this pressure issue to occur every time I work with sample containing urea. Is it a normal occurence? What can I do to prevent / minimize this?
Is there some sort of guard columns that can be used? I heard guard columns are usually used during HPLC to help eliminate impurities and was wondering if such things can also be applied for IMAC.
Any suggestions would be very helpful to me.
Thank you!
Best,
Lieke
So the column is running with a buffer containing urea at 8 M and the pressure is fine? If so, the sample must have undissolved material. Centrifuge for 2 min a 21,000g. The inlet filter will still get clogged after several injections. You can always remove it and clean it with an ultrasonic bath.
Here's a suggestion. Instead of lysing the cells with the high-pressure cell in a lysis buffer containing 8 M urea, leave out the urea but include DNase and MgCl2. This will allow the DNase to degrade DNA, which should reduce the viscosity and allow the inclusion bodies to be pelleted by a relatively low-speed centrifugation. Next, wash the inclusion bodies with a simple buffer. Then, dissolve the inclusion bodies thoroughly in 8 M urea and load them onto the column. (6 M guanidine HCl may work better than 8 M urea.) The difference from your procedure is that the DNA is broken down and washed away, so it will not contribute a high viscosity to the inclusion body solution.