After lysis, my supernatant has a light brown color to it. I’m wondering if this is common for others as well. I’m worried about this because it interferes with protein quantification, and wondering if it’s my lysis procedure or something encountered by others.
My lysis protocol (starting from a liquid culture, all resuspensions in 0.1 M ice cold NaCl):
Purify worms by:
Pellet worms (1 min @ 1000g)
resuspend in 25 mL NaCl + 25 mL 60% Sucrose (so 30% final Sucrose)
spin 5 mins & 900g
take top slurry (~ 15mL) into new tube
Resuspend to 50 mL with NaCl & pellet for 1 min @ 700g - repeat x3.
Then to lyse:
Divide worms in microcentifuge tubes, spin down and resuspend in 9M Urea + inhibitors (phosphatase/protease inhibitors)
Sonicate to break cuticles & cells
Spin down @ 16,100g for 20 mins
Take supernatant as protein component.
The supernatant is what is brownish though, which interferes with a bradford assay’s accuracy. I want to evenly mix different worm strains for MS analysis – thus having accurate protein concentrations is crucial. Any recommendations/suggestions/reasons why the sup is brown?