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Elution problem in Protein G column - Guide me (Aug/14/2008 )


I am trying to purify my protein which is tagged with FC part of Ig1 antibody, using Protein G column.But after eluteing with 0.1MGlycin Buffer PH 2.8, i not able detect protein in any of my elutes. but able to detect in beads and wash fraction.

I dont know what is going wrong...

I using 20mM Sodium phoshphate as binding buffer
0.1MGlycin Buffer PH 2.8 as elution buffer...

Need suggestion


You might want to try out this Immunoprecipitation Protocol with Protein G


your pH may be just a little bit high for elution. the affinity chromatography handbook (ge healthcare) states:

"Most immunoglobulin species do not elute from Protein G Sepharose until pH 2.7 or less.
If biological activity of the antibody or antibody fragment is lost due to the low pH
required for elution, try Protein A Sepharose: the elution pH may be less harsh."

try reducing your pH to 2.5.