Trouble Isolating Protein from Bone marrow derived-neutrophils

Hi all,

For the past 2 weeks, I have not been able to extract a significant amount of protein from my neutrophils. I use the BCA assay to quantify my protein concentration which always ends up being very small (evidenced by no color change), however, my standards are showing the right colour change. The others in my lab have no problems extracting proteins from their cells so I'm starting to think that my technique or protocol is the problem. Or maybe different solutions/buffers are required for neutrophils?

Here is my current protocol:

Note: neutrophils have been incubated overnight in a 24 well plate (~600,000 cells/well with ~1 mL of RPMI 1640 media)

  1. Place the cell culture dish on ice. Aspirate media and perform two washes with ice-cold PBS (note: cells are still adhered to the well at this stage)

  2. Add 120 uL of ice-cold RIPA buffer and 0.6 uL of PMSF (to dilute 200x PMSF to 1 mM) to well. Leave on ice for 5 minutes.

  3. Aspirate lysate into pre-cooled microcentrifuge tube. Add PBS and scrape remaining cells or lysate off the dish and add to tube.

  4. Centrifuge at 8000g, 10 min, 4 degrees.

  5. Aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.

I would appreciate any kind of advice you all can provide. Thanks a ton!