Hello,
I am trying to isolate nuclei from HEK293T cells expressing a protein that aggregates in the cytoplasm, however when I run my samples in western blot the cytoplasmic protein is strongly present in my nuclear fraction probably due to the presence of detergents. I have tried 2 different kits so far with no luck. Any ideas of a possible way to avoid this contamination? Thank you.
Have you tried increasing the washes with the Cytoplasmic Extraction Buffer ? You can increase NP40 upto 0.075%. There isnt much you can do to avoid cytoplasmic protein contamination besides increasing volume of the buffer and the washes.
If your strongly expressed cytoplasmic protein associates with the membrane fraction, it will be harder still. but passing through a 25G needle helps to further rupture the cell membranes while keeping the nucleus intact.
This is a protocol that i use :
Nuclear extract can be prepared according to the method of Schreiber et al (Schreiber et al., 1990). Cells at a concentration of 1 x 106 cell/well are pre‐incubated with or without test compound. The cells are then washed with PBS, dislodged and pelleted by centrifugation and resuspended in the cell lysis buffer [10 mM HEPES; pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), 0.5% Nonidet‐40 and 0.5 mM PMSF along with the protease inhibitor cocktail (Sigma)] and allowed to swell on ice for 15‐20 min with intermittent mixing. Tubes are vortexed to disrupt cell membranes and then centrifuged at 12,000 g at 4°C for 10 min.
The supernatant is stored at ‐70°C till further use as cytoplasmic extract.
The pelleted nuclei were washed thrice with the cell lysis buffer and resuspended in the nuclear extraction buffer containing 20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF with protease inhibitor cocktail and incubated in ice for 30 min.
Nuclear extract is collected by centrifugation at 12,000 g for 15 min at 4°C. Protein concentration of the nuclear extract is estimated using Bradford’s reagent (BioRad, USA). The extract is either immediately used or stored at ‐70°C till further use
- Badges
- Science method