Trying to measure protein concentration for Lucigenin assay. But even culturing at more than 80% confluency, after centrifugation and sonication getting only 330ug/ml of protein. Hard to maintain 100ug of protein/well.
Using Krebs-Ringer-HEPES buffer (for lysis and reaction). Not sure if it is working well as reaction buffer or not.
Need suggestions and more discussion.
Thank you.
How many are you using? Normally I utilized 5x106 of cells for protein extraction. And it can get a good yield.
Prepare a double detergent or triple detergent lysis buffer. Use 1ml lysis buffer per 10million cells (estimate your cell count before you proceed).avoid sonication if you going for a triple detergent lysis buffer 30mins incubation at 4°C with occassional tapping would be sufficient for isolating whole cell lysate (dnt forget to use protease inhibitor). You're good to go with centrifugation and protien estimation (BCA OR Bradford, whichever you find convenient.
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