https://www.bio-rad-antibodies.com/static/2015/proteus/protein-a-handbook.pdf

https://www.abbiotec.com/sites/default/files/lp109-protein_a_antibody_purification.pdf

http://www.bowdish.ca/lab/wp-content/uploads/2012/10/Protein-A-Sepharose-purification-of-Antibodies.pdf

There are many protocols. I am just going to provide some things to keep in mind. Avoid reducing compounds such as DTT and beta mercaptoethanol since these will destroy the antibodies. You can use physiological pH or one where your desired protein is more stable. Elution can be done with several chaotropic agents such as urea, low pH, guanidinium hydrochloride and others. Keep in mind that the more harsh the chemicals used to elute the protein from the antibody, the higher the likelihood that you will denature the antibody as well and perhaps render your column useless for another round. good luck.

Thank you Gustavo Zardeneta for the direction. I see different recipe in the internet for the same resin but with different protein. Do that mean the best buffer must be chosen empirically?

Hi,

first of all, keep in mind that protein A will only bind IgG . All other antibodies will elute in the flowthrough. On what refers to the buffers, PBS, TBS or tris at a pH between 7 and 8 usually work fine. For elution I use 100 mM glycine pH 3. Collect 1 ml per tube and, prior to purification, add 100 microliters 1M tris pH 8 to each tube to neutralize the acidity of the fractions. Pool the bound fraction and dialyse (or dessalt) with PBS .

good luck,

Patrick

Hi

Binding of protein A has well documented for IgG.binding buffer works better with 20mM sodium phoaphate pH 7 or 50mM tris buffer pH7 and elution buffer with 50mM glycine,pH 2.8.neutralization of fraction with 1M tris-Cl pH8.

Hi

I think the following link will be helpful for you.

https://www.abbiotec.com/sites/default/files/lp109-protein_a_antibody_purification.pdf