Hi, I have been doing some immunoprecipitation (IP) work and no matter what I do I cannot get rid of the IgG heavy chain that I see on the western blot. I am blotting for an antibody called XRCC4 which is at 55 kDa, the IgG heavy chain is around 50 kDa and it completely masks everything up. I have bough Clean-Blot™ IP Detection Kit (HRP) which on paper should have helped. However, it has only partially cleaned it up some of the lanes. Would anyone have any further suggestions I could use.
Wolfgang Schechinger
Covalently immobilize the antibody used for the IP, e.g., on carboxy activated magnetic beads or on NHS activated sepharose.
0 votes
0 thanks
Ketil Winther Pedersen
Hi,
2 ways possible here as far as i can see:
- covalently conjugate the capture Ab to the surface of magnetic beads - such as the Dynabeads Ab coupling kit
- use non-covalent Ab binding to beads such as the Dynabeads Protein A/Dynabeads Protein G or DynaGreen Protein A or A/G. here the Abs will be eluted off but if combined with TrueBlot HRP labeled Sec Ab for WB - they will not recognize heavy or light chain Abs on the blot.
kind regards
ketil
0 votes
0 thanks
- Badges
- Science method
More Berfin Barlas's questions See All
Similar questions and discussions