Issue with separating cytoplasmic and nuclear extract without contamination - (Sep/10/2007 )
I have been trying to collect cytoplasmic and nuclear extract using the usual hypotonic + detergent / hypertonic treatment (the recipe is somewhere in this forum), then I use Western blot to see if they are contaminating each other.
1. I can't seem to find a good antibody for nuclear protein, so my result always shows cytoplasmic protein contamination. ph34r.gif (I've tried p-53 and creb-1). Anyone know of a good clean antibody for nuclear protein?
2. I use Gapdh to check on my cytoplasmic extract. I've tried using the lowest amount of detergent to lyse the cell, but I'm still having nuclear contamination. My guess is that the detergent may be too rough on the nuclear membrane (NP-40)? blush.gif Any recommendation?
Thanks guys. =)
generally 0.05% NP40 is minimum routine for lysing cell membrane but not nuclear one.
So use this concentration. Also, don't over-pipet your first supernatant ! nuclear pellets are not a very hard pellet. so over pipetting may be sufficent to contaminate.
We enhanced our nuclear protocol extraction. After cell lysis, we wash the nuclear pellet with a bunch of A buffer without NP40.
Transcription assays are truly enhanced !