IP FLAG Phusion protein - Lysis conditions (Jul/27/2005 )
thanks for answering.
The problem is I cannot use NP-40 in the lysis buffer.
I have always used NP-40 in my lysis buffers but this time the protein is 26-27 kDa size , and that is too close to the light chain of the antibody
reason why I am now using affinity gel (FLAG antibody attached to agarose)to immunoprecipitate FLAG phusion protein.
NP-40 is not compatible with the use of this gel/beads as because it can result in the dissociation of the antibody from the agarose beads.
According to manufacter instruction I can use 1% Triton
that is why I ask:
Is Triton X 100 as good as NP-40 to get nuclear protein?
Thanks once again
i use NP40 for extracting cytoplasmic and nuclear proteins, even if these extraction are realized for total extract or for an extract that separates cyt prot from nucl prot.
works very well.
Triton is more stringent than NP40, so in principle you shouldnt have any problems with extraction of nuclear protein. 1% Triton extracts more of the stuff, and actually I use low NP40 conc. to keep nuclei intact during cell lysis, whats rather not possible with Triton.
However if you have any reason to stick to NP40, I've been using even up to 1% NP40 during FLAG ips with affinity gel. No problems with the beads. Best is to check how does it look in your conditions.
However using FLAG beads you still need to be careful. Ab in the beads is much "concentrated". You'll suck up "one bead" during the final IP collection, and you'll end up with heavy chain anyway. Also, I always prepare beads by glycine wash to remove unbound Ab.