No :3 affinity colum  would be your best bet, or you can use antigen specific speros beeds to purify your Ab. let me know if this helps

Test bleeds usually have not enough volumes for sufficient antigen affinity purification. Do you need to test for picking the best rabbit, then the serum should probably suffice for a first impression. If it is a matter of testing subsequent bleeds, you may have missed the optimal titre by now anyhow. First test the sera, then pick the best rabbit and go for full scale antigen-affinity purification (no.3).

They are the steps I use for my large scale antibody purifications - in many of my assays total IgG (Just protein G purified) antibody suffices. For a test bleed you should be able to test whole serum, and then do a Prot A/G purification from there if needed for your application. However antigen purfiication may not  yield much unless your antibody is in very high titre in serum. If your application is for western blot/ELISA do a test run with whole serum - if you don't see high background or 'non specific' binding you may not need to purify your antibody at all.

Thanks all. I tried WB with two subsequent test bleeds (not purified) and I see a similar pattern for both the pre and the test bleeds. I also see other bands and a band at the area where we expect for the protein of our choice. So, now I don't know

1) If it is specific at all.

2) Maybe there is another band that is close to our protein of interest that is non-specific, so it shows up in both.

I want to test if it is worth continuing with this immunized rabbit or not. So, I wanted to purify and then test it using a WB. 

Is there a reference(s) that would give me information about this whole process of selecting the Antigen, selecting the animal, test to perform to know if the Ab is specific, that any of you is aware of ? 

Dear Sivasankari , in agreement with above answer, I will choose Protein A/G affinity chromatography to purify IgG from rabbit serum in this scale. Purification protocols based on differential of solubility (e.i salting out with ammonium sulphate) are more adequate for large volumes of serum. Bioaffinity chromatography is an excellent option but you must have enough antigen to coupling to a matrix (mgs). On the other hand, it could be "the tale of the egg and the hen". If you dont have pure antigen all antibodies that you obtain will be against all proteins  immobilized onto  the matrix.

I understood that you want to use these polyclonal rabbit antibodies in a WB and in accordance with Natalie´s suggestion, you assayed complete serum in WB. Many bands were observed in this assay in both immunized and non immunized rabbit sera including the protein band of interest. Am I right? If yes, I question you, why non immunized animals have antibodies titer vs your Ag? What is the serum dilution that you are using? What  kind of antigen are you using?

Thank you Oscar Otero for taking the time to write. Here are my answers:

Many bands were observed in this assay in both immunized and non immunized rabbit sera including the protein band of interest. Am I right?

yes.

If yes, I question you, why non immunized animals have antibodies titer vs your Ag?

That's exactly why I want to try a purification. Reasons that I think : 1. The Ab did not work and all I see is the reactivity of the Ab from the sera to a bunch of proteins in my lysate. 2. There is a non-specific Ab that reacts to my protein of interest/protein of closer molecular weight, so it ends up showing up in both the pre and the test bleed. 3. The titre of the Ab is less. 

What is the serum dilution that you are using?

I used 1:1000 and 1:500. 

What kind of antigen are you using?Add your answer

Recombinantly expressed N-terminus of the protein for which we are trying to make Ab. 

Hello Shivasankari,

I would not recommend going through the process of purifying antibody.  Based on what you have described, it would seem that the immune serum you have will not be selective enough afterwards, unless you are able to do an affinity purification, where the solid phase of your column has the N-terminal polypeptide.  If this was a small expression, I am curious, what was the carrier for antigen?  KLH, BSA?  I ask because if your cell lysates are being lit up by both pre-immune and immune serum, then something has gone wrong.

If the rabbit has antibodies to components of your expressing cell line, then it is likely not ever going to produce satisfactory antibody, and you should either switch cell lines for expression, or choose a different animal, such as goat.

I say this with experience testing and creating and having antibodies produced across species, both with crude antigen as well as polypeptide+carrier scenarios.

Stephen, I am not sure what the carrier was. I am indeed planning to do an affinity purification with the Ag that was used to immunize the rabbits. My hopes are not too high too, but just want to make sure if this works or not for all the money and time spent on this. I will try to know what the carrier was and it is an interesting thing to look into. Also, what would I know by knowing the carrier ? 

Thank you for the valuable inputs. 

The carrier protein, especially when BSA is used, will cause the injected animal to produce anti-BSA Ab's, and because of its widespread use in buffer solutions, can create a reactive Ab set.  KLH doesn't pose that type of problem generally, but the conjugation requires more than with BSA, so BSA is generally used unless you specify for it not to be included.

The pre-immune serum, especially with WB, just should not light up bands.  Any chance your protein of interest has a homolog in rabbit? 

Thanks so much. Looks like a carrier was not used. 

Thank you so much for invitation but this topic is out of my research field so I can not post a scientific comment.

Regards, Emad