I need perform western blot for HAEC cells but the total protein I am extracting is not enough for it. I am using 75 cm² flasks and, according with my experience, this amount of cells should be enough for WB. How can I improve protein extraction from these cells?
Some proteins tend to disappear if subjected to std. SDS-PAGE conditions, notably some complex membrane proteins, but there should be plenty of other proteins that you can see with Ponceu S before going on with blocking/Ab staining. You need to check all the steps, protein in extract, extract transfers to membrane, Ab recognizes proteins on membrane.
Some antibodies are just weak, you might consider blotting an internal control like b-actin.
I agree with Andrei Nikonov but I suggest to add some proteases and phosphatase inhibitors to your lysis buffer or otherwise you can use the RIPA buffer instead the water.
I use a different kind of protocol for protein extraction. I wash the cells in PBS. Detach the cells from plastic with cell scraper with PBS. Centrifuge at 2,500 g for 5 min at 4C. Discard supernatant. Add 1 volume of RIPA to the cell pellet, suspend and vortex. Then I usually perform a step of sonication in a ultrasonic bath. I centrifuge again for 10 min and I recover the supernatant. I quantify it with BCA or Bradford and then I prepare my samples adding Leammli Buffer + beta mercaptoethanol. I boil them at 95C for 5 min. Everything must be performed in ice and in a centrifuge at 4C and obviously there are proteases and phosphatases inhibitors in my RIPA buffer.
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