1 reply to this topic

[nycmerckx]

Hi,

I am trying to IP a FLAg tagged protein. I can detect my protein of interest and proteins that should bind it by western. I want to use the IP to do MS of the binding partners.
I lyse about five 10cm dishes and get about 5mg/ml of protein. I then use about 2mg of that protein and incubate it with FLAG antibody overnight at 4C. I sue a 1/50 dilution of antibody. I know I should state ug quantities but I dont have the info. I also think the problem lies elsewhere. I then add 50ul of agarose A slurry to the antibody/bait mix. I incubate this for 2 hrs at 4C. I wash 3x. I then boil in laemlli buffer and run a SDS PAGE. I stain using colloidal blue which can detect down to 15ng of protein. I get nothing.

So its detectable by western and not by colloidal blue which has me scratching my head. How do I get a stronger signal on my coomassie?  should I be using more protein in the initial antibody trap reaction?  should I use FlAG M2 beads?  please help this is grinding me down.

[polyfractal]

I'm not an expert in this field, but 5mg/mL protein content seems a bit low.  When I collect lysate under similar cell culture conditions and nanodrop, I routinely get 15-20mg/mL.

Try reducing the amount of lysing buffer you are using or use more protein in the antibody bait step to get a higher concentration.  Perhaps your agarose A slurry is old and no longer as effective?  Also make sure that Protein A is best suited for your particular antibodies and host.  Protein G may be better.  Consult this PDF (second page) for a list of binding strengths:

http://www.kpl.com/d...cs/PURIFIGG.PDF