When lysing E. coli for recombinant protein expression, I utilized a lysis buffer composed of 1M Tris-HCl, Triton X-100, glycerol, lysozyme, MgCl2, and a protease inhibitor cocktail. After lysing the E. coli, I centrifuged it at 13000rpm for 1 hour and collected the resulting supernatant. My intention was to pass this supernatant through a 0.45μm cellulose acetate syringe filter to eliminate any remaining cell debris. However, the filter became easily clogged. I'm wondering which components might be causing this issue. Could it be the conditions of centrifugation being too mild, or something else? The volume to be centrifuged in each tube is typically 200mL.
All filters will get clogged after a few mL of supernatant have passed through, better results are found with lateral ultrafiltration. Everything contributes toward clogging of the filter: the presence of small cell debris, the presence of precipitated proteins, protein polarization, protein adsorption, etc. You can increase the volume that can pass through a single membrane, but you will eventually clog it. Centrifuge at higher relative centrifuge force before filtration.
It would also help to add some DNase to break up the DNA and thereby lower the viscosity.
But, after the lysozyme reaction, I subjected the mixture to 30 minutes of sonication. So, when observed visually, it appears to have low viscosity. Are there any factors other than viscosity?
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