Role of buffer in antibody purification - (Dec/30/2005 )

what is the role of each chemical function in antibody purification
1.binding buffer(sodiumazide,glycine&nacl)
2.elution bufferb1(sodiumazide&citricacid(anhydrous&trisodiumsalt)
3.elutionbufferb2(glycine&sodiumazide)

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I'm not sure i can help you with the chemicals but they are surely related to the kind of matix that you use to make the isolation of the Ab. Na-azide is generally used to avoid microrganisms growth but I don't now if it plays other functions here.

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Binding Buffer has a higher pH than the elution buffer. Ab in that particular pH efficiently binds to the beads.
Elution Buffer has a lower pH than the binding buffer. There are a number of elution buffer with different pH, as different isoforms of Ab (IgG1, IgG2a, IgG2b, IgG3) eluted at different pH.

Besides, different column may use different binding buffer. Protein A column use Glycine/NaCl pH8.5 and Protein L column use NaP/NaCl pH7.2 etc.

I think it is the pH of the buffer that is important. Some buffer is more stable in certain range of pH than others.

I hope this may help.

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hello,

to purify antibodies, you use a matrix where protein A or protein G is bound.
It will bind the Fc part of the antibody.

with protein A the appropriate pH for binding is pH 8.0. I use PBS pH 8.0.
to elute the antibody, you need to reduce pH. it depends on the antibody you want to purify.
for example : you will elute mouse IgG1 with pH6.5, IgG2a with pH4.5, IgG2b and IgG3 with pH 3.0.
I am using 0.1M citric acid at suitable pH.

It is recommended to quickly neutralize the pH of the eluted antibody and the column for their stability. I use tris.

you can also use protein G. It's better for some human and rat antbodies.
binding and elution are done at a different pH than for protein A.

hope it will answer your question

good luck

laurence

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