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protein purification from agarose beads using immunoprecipation kit

protein purification anti-myc agarose beads

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#1 ajitk76

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Posted 16 January 2017 - 07:19 AM

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 



#2 mdfenko

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Posted 16 January 2017 - 11:44 AM

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.


talent does what it can
genius does what it must
i do what i get paid to do

#3 mdfenko

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Posted 27 July 2017 - 05:09 AM

that link doesn't work. try this one:

 

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


talent does what it can
genius does what it must
i do what i get paid to do





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