Affinity purification is more complex as you may expect. There are many things that can go wrong.

1. Why do you want to purify your serum? If you have a bad serum, this will not help you. Perhaps a simple purification with Protein A or G will be sufficient. Other researchers recommend a subtractive approach (remove unwanted reactivities), instead of an affinity purification.

2. If you have a good serum, the antibodies of interest might bind too tightly to your antigen. Then the glycine buffer will not be able to elute a non-denatured antibody. If you would use a weaker eluent, the antibody/antigen complex might not dissociate.

3. Usually, you have to optimize the pH of your glycine elution buffer. Useful pH can be between 2.0 and 4.0.

4. Affinity purification on a Western Blot leads to very small amounts of purified antibody. Affinity columns or magnetic beads might lead to much higher amounts.

5.Did you check the amount of eluted IgG? Perhaps your concentration (or yield) is much too low.

Yes. Your protocol is correct. You need to obtain brand new unexpired reagents and only use those. Then make sure they are stored correctly and that only you have access to them so there is no chance of contamination or degradation. That is usually the problem :-)

Affinity purification is more complex as you may expect. There are many things that can go wrong.

1. Why do you want to purify your serum? If you have a bad serum, this will not help you. Perhaps a simple purification with Protein A or G will be sufficient. Other researchers recommend a subtractive approach (remove unwanted reactivities), instead of an affinity purification.

2. If you have a good serum, the antibodies of interest might bind too tightly to your antigen. Then the glycine buffer will not be able to elute a non-denatured antibody. If you would use a weaker eluent, the antibody/antigen complex might not dissociate.

3. Usually, you have to optimize the pH of your glycine elution buffer. Useful pH can be between 2.0 and 4.0.

4. Affinity purification on a Western Blot leads to very small amounts of purified antibody. Affinity columns or magnetic beads might lead to much higher amounts.

5.Did you check the amount of eluted IgG? Perhaps your concentration (or yield) is much too low.

Hi Babuta.

My name is Oscar Otero and I work in the CIM (Center of Molecular Immunology) in Havana, Cuba. I saw your question in RG and I tried to answer it but for unknown reason I couldn’t sent my answer by RG. So I sending my opinion in this way.

First, I am in agreement with Michael's comments. I ask you: Are purifying Ab via WB for use it in WB? If yes, I don’t understating why you are doing this.

If your goal is identify some antigens in a mix, you can use the crude serum without any problem. Only you have to select the correct dilution and an appropriated conjugate to develop the reaction. Try 1/100 to 1/1000 dilution of the serum.

I think that's not good idea elute an Ab at extreme pH and after that, use it in a WB assay. On the other hand, WB in not a purifying technique!! Maybe you can obtain very little Ab through this procedure and it will be not enough for detect any band. I suggest you use affinity chromatography using some Protein A or G matrix. Of course, it will depend of the origin of your Ab, but Ab coming murine, rabbit, human and very different mammalian species are easily purified by this way. Amounts enough can be obtained using this procedures. Please, if you need any additional suggestion, you can send me a personal message. My email is [email protected]. You can publish my answer in RG too, because I can't do it, I don’t know why. Best regards, Oscar.

Dear Michael,

Thank you for all the suggestions.

Detail of my experiment, I have generated antibody in mice in our in house animal facility. Its a good sera as I could detect my antigen of interest at a dilution of 1:1000 in first booster to 1:4000 in fourth booster. The problem is, there is two isoform of this antigen (70 nd 55kDa) and I want to get rid of the second one. I want to use this purified antibody in western blot. 

I eluted the antibody using 0.2M glycine pH 2.2. I directly boiled a bit of  PVDF membrane and could see the antibody still bound to it, however the eluted antibody yield is much less. 

 can you suggest any alternative protocol my email id is [email protected]. 

Thank you for your additional information. I have still some questions left...

1. Why did you use a mouse for immunization? The amount of serum is very small from such a small animal. Any purification will lead to very small amounts of purified antibody.

2. How did you immunize the mouse? Do you have the pure 70kDa form?

3. How did you perform the affinity purification? Did you use the 70kDa form, too?

4. If your 55kDa form is a fragment of your 70kDa form, you would need a very specific affinity purification anyway. Do you have the purified 55kDa form available?

5. Why do you want to get rid of the second band? This might indicate the same epitope and hence no "non-specific" reaction.

Hi Michael,

answers to your question

I immunized the mice using smaller fragment of 70kda which is unique to this protein and used the above mentioned smaller fragment to affinity purify the antibody, unfortunately I donot have 55kDa isoform in expression vector. 

Do you know the sequence of the unwanted 55 kDa fragment? If yes, does this sequence overlap with the sequence of your immunogen?

I think the questions above about the similarity of the 55 and 70KDa forms are highly relevant to the purification strategy, but I would like to share an experience I had of using blots to purify polyclonal antibodies. I used a 'single track' gel. I don't know if you can buy such a thing now (I had to make my own at the time) but you could also load a standard gel (with multiple tracks) with just one sample if necessary. Run the gel and blot and then stain the blot with Ponceau S dye. Cut the blot horizontally so that you have a thin strip containing the protein band of interest. Block the strip with TBS/BSA (this will also destain the strip) and then incubate with the polyclonal serum. After washing, suspend the strip vertically and treat it like a 'column', adding 50ul portions of acid at the top and allowing the liquid to run down the strip. This keeps the elution volume very low. You can collect 100 ul fractions into eppendorfs and neutralise, just as you can with columns. I had great success in purifying antibodies to subunits of a multi-subunit protein immunogen. I do not think the acid elution is likely to be a major issue with a polyclonal Ab, as long as you neutralise quickly. Indeed, if there is an issue of loss of high affinity antibodies (a theoretical weakness of this approach) you can always further elute your 'column' with acid of even lower pH e.g. 2.0 and then 1.8 if necessary. If the antibodies are stuck on the membrane anyway, you have very little to lose. 

Thank you Michael I have checked the sequence and there is no overlap as such.

Thank you Nick for the suggestion I will try this way.