Anyone have a protocol for making cell lysate from rat cardiomyocyte pellets to run an ELISA using PBS as the buffer?
Hello Eric Yalley
You may follow the protocol provided below (via freeze-thaw method).
1. Re-suspend the cell pellet in PBS containing protease inhibitors to prevent proteolysis of protein of interest during cell lysis. Upon cell lysis, endogenous proteolytic enzymes are released from subcellular compartments which would degrade the protein extract before analysis for targets of interest. A mixture or inhibitor cocktail of several different protease inhibitor compounds bind to proteases ensuring that there is no degradation of protein of interest.
2. Freeze the cell suspension using dry ice/ethanol bath or freezer for 10 minutes.
3. Thaw the cell suspension at room temperature or in a 37 degree water bath.
4. Repeat the freeze-thaw cycle, 3-5 times.
5. Remove cell debris by centrifugation at 13,000 rpm for 5-7 minutes.
6. Use the supernatant to estimate the total protein concentration of cell lysate by BCA assay. This would be necessary to normalize the volume of sample that can deliver the same amount of total protein for ELISA. Usually, total protein concentration of cell lysate should be at least 1mg/ml.
7. Perform ELISA.
Best.
@Malcolm Nobre how much PBS should I be using to suspend the cell pellets
Eric Yalley use 3-4 million cells in 100ul PBS. What is the cell count?
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