I was wondering whether anyone has worked with H9C2s and could recommend a cell lysis buffer or protocol. This cell-line is still new to me and I am currently optimizing a cell lysate protocol. My last troubleshooting run yielded very low protein concentrations and I'm not sure whether this is related to my protocol or the buffer used (which was RIPA buffer). Any advice would be greatly appreciated.
Hi Sam, try with this protocol
Extraction of protein
H9c2 and H9c2T1 cardiomyoblasts were harvested, homogenized in lysis buffer (50 mM Hepes, 200 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween-20, 10 mM βglycerophosphate, 1 mM NaF, 2 mM Na3VO4, 1 mM DTT, 0.2 mM PMSF, 5μg/ml leupeptin) and incubated on ice for 90 min with intervening vortexing. After centrifugation of the homogenate (15 min at 14000 rpm), the supernatant was
recovered and stored at -20°C.
For more information please have a look in:
http://www.diss.fu-berlin.de/diss/servlets/MCRFileNodeServlet/FUDISS_derivate_000000002779/03_kap3.pdf?hosts=
Gook luck
Hi Natasha,
You can make concentrated protein lysates by reducing the amount RIPA buffer you use per given number of cells or by increase the number of cells you lyse in a given volume of RIPA.
Best Wishes
Thanks Natasha / George, I'll try out the recommended buffer in my next run.
Hi,
Have you tried with Hepes buffer? I've experienced the same problem as you did. Wondering if you successfully troubleshoot it.
thanks!
Hi Darren,
I used the recipe recommended by Buchs and I've been getting good protein yields so far. In my last run I was able to get yields of > 1 microgram per microliter for each well which is enough to run westerns. I seed my cells in a 6 well plate and add 150 microliters of lysis buffer into each well. If you have any other questions let me know.
Good luck!
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