I'm attempting to concentrate whole cell lysates from primary murine cells, in order to load enough protein onto a gel to detect my protein of interest. I'm pretty new to protein purification and western blotting so any tips you can provide would be much appreciated.
During my lysate prep, I incubate the cells on ice for 30 minutes in Tris-buffered 1% SDS, 1% Triton X-100 containing a cocktail of protease inhibitors. I then sonicate and freeze/thaw to shear DNA, and clarify the lysate by spinning at max speed for 10 minutes in a table-top centrifuge. Usually I'm able to achieve a protein concentration around 0.5 mg/mL, and the solution is not viscous.
I then transfer the solution to an Amicon 10 kDa cut-off spin filter, and spin in 10 minute intervals at max speed, resuspending with a pipette between each interval. By the end I've usually reduced the volume and increased the protein concentration by a factor of ~15, but the solution has become VERY viscous-- about the consistency of Triton X-100, I would say. When I try to run this very viscous solution on a gel, I get badly misshapen bands.
Could I actually be concentrating detergent? I know at a certain concentration, detergents are more likely to form micelles which are bigger than 10 kDa. How should I get around this if this is the case? The other thought I had was that DNA shearing might've been incomplete. Should I try more sonication and/or freeze/thaws?
Thanks for any help you can provide!
Hello Sean,
I am surprised that you should get only 0.5 mg/ml protein in your primary extract. As a first measure, I would suggest that you decrease by a factor of 10 the volume of lysis buffer that you add to your cells. Thus you won't need to reconcentrate your samples by ultrafiltration (and save a significant time and money). It is quite likely that the viscosity of the reconcentrated samples is due to concentration of detergent, which forms micelles. If DNA is a problem regarding viscosity, adding some Benzonase or the equivalent DNase supplied by Pierce will save you the time of sonicating the samples. Of course the DNase will appear as a band with a size of ~ 30 kDa in your samples, hence it is advisable to run a lane with it next to your treated samples.
Good luck,
Pierre
Hi Sean,
While they are probably related, you could be dealing with two separate problems: 1) The lysis and filtration protocol giving you a viscous sample and 2) Misshapen bands on western blot. A sample issue will cause problems in the following western blot - but even if your samples are good, you could get misshaped bands. Maybe you can include control samples (lysates which previously yielded good bands) next time, just to ensure the problem is with the samples and not your western blot protocol.
First I would follow Pierre’s advice; lowering the volume of initial lysis buffer (or increasing the number of cells/size of pellet) will increase the concentration of the primary lysate, hopefully enough to prevent the need for filtration. If you still need to concentrate your samples, however, you can try lowering the concentration of detergent; say 0.1% TritonX (as well as lowering SDS to 0.1%). Check if the protein yield from the two buffers is roughly similar, and then try concentrating the samples on filters and see if viscosity changes with lower detergent concentration.
Are you sure you need to resuspend and filter over several intervals? When I’ve used Amicon ultra filters in the past, I ran whole samples (500 ul samples, the small filters) at 13’000 rpm for 10-15 minutes and this would yield approximately 50 ul concentrated lysate. I’m wondering if continuously resuspending with lysis buffer (if that's what you use) could cause a pool-up of detergent/triton in your final sample.
Best of luck!
Sean Simpson
As others have mentioned, try to keep your resuspension volume as low as possible, because its always easier to dilute further than not having enough. Another thing is, if you are experiencing viscous lysate, you can add BME, PMSF, DTT, to your loading dye and heat the sample (95C) before loading on the gel or use a more potent lysis buffer (I always use a Urea buffer for protein lysates).
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