2 replies to this topic

[ajitk76]

Hi,

 

We are using immunoprecipitation kit having anti-myc-coated agarose beads. however, the kit does not provide elution buffer for purified protein. We would like to elute purified protein for further use. We tried to elute with 0.1 M glycine buffer (pH 2) and neutralize with 1 M Tris buffer (pH 8). Initially, we use glycine buffer for 5 to 10 min. but protein was not eluted. Further, we increase the time to 20 min. then it vanishes in Western blot. Please let us know the protocol for glycine buffer elution (time and quantity used and temperature condition etc for glycine use and same for Tris-neutralization). Thanks,

Ajit

ajitkul1976@yahoo.com / ajitakulkarni76@gmail.com 

[mdfenko]

you should probably use pH 9 tris in the collection tube (have you verified neutralization?).

 

you can let the buffer sit in the co;umm for about an hour to maximize elution.

 

you can try pH 10-11 to elute (with pH 6 neutralizer).

 

i recommend that you download the "Affinity Chromatography Vol. 1: Antibodies" from this ge lifesciences webpage.

[mdfenko]

that link doesn't work. try this one:

 

http://www.gelifesci...port/handbooks/