Hi everyone,

I really don't know how to fix the issue that I am facing.

For background, I am working on the purification of inclusion bodies protein, which require the use of 8 M urea in the buffer (we do not use guanidine in this case as it is way more expensive and we don't have the budget *yes I know :( )

Anyway, back to my issue... I did the E. coli cell lysis with osmotic shock, wash the pellets with Triton X-100 and 1 M NaCl, and did filtration after centrifugation (with paper filter, as vacuum filter did not really work well). So during loading everything was fine except the pressure increasing, which I thought was normal. However, when we started the wash no liquid (or in very small insignificant amount) came out of the waste. The pressure was also not decreasing no matter how long we waited.

So we thought something was wrong with the machine, which is ÄKTA start (it does not have prefilter). We took out the columns and tested all the tubings and pathways, and I found out no liquid is coming out from the conductivity sensor. Thus almost none goes to the waste. Since it's Friday, we run the column pathway with 2 liter of pure water at 0.5 mL/min (that's the slowest flowest rate the machine can do).

This morning (on Monday) I found out only 400 mL water is used from the full 2 L water we prepared. There is water coming out, but still not at the right rate and the pressure is still high 0.37 MPa. I compared it if the pathway goes directly to waste, and the previous is slower. So something is still wrong.

Can anyone offer a solution? I really have no idea. The main issue I think lies on the conductivity sensor, which unfortunately cannot be opened. Also for info, after every run I clean the columns and the machine with 1 M NaOH.

Kind regards,

Lieke