I'm new here but have been using this site as a resource for a while, and now I have a question to ask regarding a problem that I've been stuck on for a few months. I've been trying to develop an immunoprecipitation protocol for isolating pannexin-1, with the hopes of performing coimmunoprecipitation afterwards.
The problem that I've been having relates to IgG contamination when I run the precipitated samples through SDS-PAGE/Western blot. I've done the following:
Incubate 5 ug of primary antibody with magnetic beads for 1 hour at room temp (wih rotation). Wash 3x with 200 ul of 1% Triton X-100 lysis/wash buffer.
Apply 500 ug of whole-brain lysate, prepared in the same Triton X-100 buffer as mentioned above. Incubate for 2-3 hours at room temp (with rotation)
Wash 3x with 200 ul of Triton buffer
From here I've tried three elution strategies:
1. Standard Western blotting sample prep: take the beads after the supernatant has been removed and boil with beta-ME for 3 minutes then load into the gel
2. Glycine elution (0.2M glycine pH 2.6 for 2x 10 minutes, pooling the supernatants and neutralizing with 1M Tris pH 8.0) under reducing/denaturing conditions (2% or 0.25% SDS loading buffer with 5% beta-ME, boil for 3 minutes)
3. Glycine elution (same as above) under only denaturing conditions (no beta-ME, 2% SDS loading buffer, boil for 3 minutes)
What I've been finding:
1. 55 kda and 68 kda bands. Given that the target protein is 43 kDa or so, is it feasible that the 68 kDa band is actually the target protein + the Fv antibody fragment (25 kDa) that was cleaved off under the reducing conditions?
2. Only a 55 kDa (IgG heavy chains) band
3. Bands at 190 kDa, which indicates the possibility of the target protein (~43 kDa) remaining bound to the IgG and migrating with it down the gel
The central problem appears to be that my antibodies (Rb anti Ms; which is also used for the Western blot detection steps) don't want to release the protein under my elution conditions. Short of cross-linking the Ab to the beads, is there some set of conditions that could be employed to separate the Ab from the Ag?