Affinity Purification of Chicken Antibodies

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Joanna_LaBresh
Joanna_LaBresh's picture
Affinity Purification of Chicken Antibodies

 We are attemptting to do a affinity purification of some IgY. We made a column using the protein. Blocked it. And then added our antibody. We have tried several elutions with no success. Does anyone know the secret? Any assistance would be appreciated.

Chin Fen Teo
Chin Fen Teo's picture
Hi Joanna_LaBresh,

Hi Joanna_LaBresh,

Can you provide the condition(s) that you have tried? It is easier to troubleshoot if this information is available.

Cheers.

Joanna_LaBresh
Joanna_LaBresh's picture
 We tried several… low pH (2

 We tried several… low pH (2.0) (glycine & citrate/phosphate); high pH  (triethylamine); high salt MgCl (switched to TBS here)

Chin Fen Teo
Chin Fen Teo's picture
Hi Joanna_LaBresh, if your

Hi Joanna_LaBresh, if your IgY is captured on the protein column, low pH should be able to elute the IgY VERY efficient. Key is you have to incubate the column in such condition for ~5 min before you start collecting the eluate.

If you have already done that, then the question is, have you followed the flow through/unbound fraction to make sure that the problem that you have is not because of the binding?