Hi everyone,

We have a problem with running lysates from Hi5 cells on our Aekta system for large-scale purifications using 1 mL or 5 mL Histrap columns.  The pressure is simply getting to high during sample loading.  We are lysing cells with a cell disruptor and add DNAse, clear the lysates by centrifugation, but we still have this problem.  It is specific to Hi5 lysates and not Sf9.  Has anyone else had this problem or have any suggestions for solving it?  We could ultra-centrifuge lysates before loading, but I was hoping there is an easier solution, as we don't have our own ultra.

Thanks!

best,

peggy

Hi Peggy,

everyhting that is going to the AKTA, must be filter with 0.45microns to eliminate particles. beside buffers, the most important one is the crude material (or lysate) that usually is a little coloidal even after longer centrifugation. I allways filter the lysate after a long centrifugation of 20minutes 11,000 rpm at 4C. The filter must be at least 0.45micron for columns as Ni columns; but at least 0.2micron for high resolutive columns as MonoQ or MonoS or similars. 

If the lysate is very colloidal, I prefilter with GF/B from Whatmann and then 0.45u. Sometimes if there is a big quantitie of DNA, the filtration is difficult; in these cases is preferible to incubate with higher DNase concentration. NEVER load spin lysates directly to the column

If you are afraid of filtration because protein lost, check a sample before and after filtration. I don't remember that I lost material during filtration; but who knows......

Mario

Hi Mario,

Thanks for the suggestion - we have tried filtering with a .45 bottle-top filter, but it doesn't work well (nothing passes through, or passes extremely slow).  How do you do your filtration?  And how much are you diluting your lysates?  Maybe we should be diluting in larger volumes.  In this case we diluted 4 L of cells (1 x 10^6 cells/mL) in 250 mL.

thanks!

best,

peggy

Hi Peggy,

When we had a similar problem, I found stirring with Benzonase (as opposed to DNase) for 10-20 minutes helped.  If the protein is OK, I do this at room temperature.  I then can get the lysate to pass through the filter.  If it doesn't go through the filter, don't load on the column!  I tried sonicating to shear the DNA, but this seemed less effective and I didn't want to sonicate too long and fry my protein.  I also tried diution, but it didn't really help.

Hope this helps

Jo

A longer DNase (or Benzonase) incubation as Jo said could help. More lysis buffer is very helpfull too. For bacterial pellet I usually use 1:10 of the initial culture volume. It means that for pellet of 4L I would use at least 400ml lysis buffer. Try to use prefilter, is a trick I learn in the industry, since it extends the filter volume capacity of 0.45u