Hello

If I understand correctly, you want to purify the antibody from the anti-serum, but you already have the purified protein (antigen) to use. Your problem is that the protein is too small plus the isoelectric point 8,5.

The only time I was doing antibody purification, I was using a simple procedure that worked fine, my protein was bigger (around 30 kDa) and that was based in a western blot protocol, so I think it could be used for your experiment. But you need a significant amount of your protein/antigen/epitope which needs to be purified previously using another tyoe of procedure.

I'll explain briefly the protocol I use for antibody purification by affinity:

Basically you load a significant amount of that protein in a acrylamide gel with one major well with the width of gel. For a 10kDa protein, you would need to use a high % of acrylamide like 15%. You run the gel, transfer the protein to a membrane and stain with Ponceau. You should see a strong lane with the MW of your protein if it is well purified. Then you cut that section of the membrane containing the lane of your protein and you cut it in small pieces, destain to remove the Ponceau and then continue as if you were doing a western blot. You block those pieces of membrane and then incubate with your anti-serum overnight. At the end of protocol and after washes, you add a solution of Glycine with low pH to dissociate the antibody from your protein and then neutralize the purified antibody solution and add BSA.

There are for sure better techniques, but this one worked for me and was very simple to execute. If you want to try, I can give you the protocol. You'd have to adjust the buffers of western blot based on your protein characeristics.

Good luck with your experiments,

Liliana

Please give us more information about your antibody. Overall:

You can use two methods:

First: Affinity chromatography with resin that contain specific antigen for this antibody.

Second:

1. Adding equal volume of acid acetic (pH 8.5) to the samples.

2. Centrifuge at 4 C and then obtain plette.

3. Then resolve this pellet in PBS.

4. Dialysis by appropriate bag.

This procedure must be repeated several times and between them, SDS-PAGE must be done to evaluate purity.

When, you find one band in SDS-PAGE, your protein is pure.

Hello

If I understand correctly, you want to purify the antibody from the anti-serum, but you already have the purified protein (antigen) to use. Your problem is that the protein is too small plus the isoelectric point 8,5.

The only time I was doing antibody purification, I was using a simple procedure that worked fine, my protein was bigger (around 30 kDa) and that was based in a western blot protocol, so I think it could be used for your experiment. But you need a significant amount of your protein/antigen/epitope which needs to be purified previously using another tyoe of procedure.

I'll explain briefly the protocol I use for antibody purification by affinity:

Basically you load a significant amount of that protein in a acrylamide gel with one major well with the width of gel. For a 10kDa protein, you would need to use a high % of acrylamide like 15%. You run the gel, transfer the protein to a membrane and stain with Ponceau. You should see a strong lane with the MW of your protein if it is well purified. Then you cut that section of the membrane containing the lane of your protein and you cut it in small pieces, destain to remove the Ponceau and then continue as if you were doing a western blot. You block those pieces of membrane and then incubate with your anti-serum overnight. At the end of protocol and after washes, you add a solution of Glycine with low pH to dissociate the antibody from your protein and then neutralize the purified antibody solution and add BSA.

There are for sure better techniques, but this one worked for me and was very simple to execute. If you want to try, I can give you the protocol. You'd have to adjust the buffers of western blot based on your protein characeristics.

Good luck with your experiments,

Liliana

Hi Gurjot, as Liliana said, your question is not clear.

I have immobilized tripeptides on Affigel-10 affinity media to purify integrins .

http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_1085.pdf

If you can, link your protein > 5 mg/ml resin. Wash the resin with buffers that have 0.5 M NaCl. Elute the antibodies with pH 2 buffers.

What I understand is that you have an antibody against low molecular weight protein having isoelectric point of 8.5. If you have high titer antiserum against said protein then simply you purify rabbit IgG by using proein A. 

Coupling your antigen protein to cyanogen bromide-activated-Sepharose 4B (Sigma) or Affigel (BioRad) at pH9.5 coupling buffer, it should be OK for your 10kDa and IP 8.5 antigen protein. Keep in mind, your antigen protein's purity should be >95% and changed the buffer to pH9.5 coupling buffer before mix it with activated beads. You can use this resin to do affinity purification of your antibody from rabbit serum.

Hi Evereyone,

Thank you for your answers.

Liliana: yes Liliana, you are right. I want to purify the antibody from the anti-serum, and I already have the purified protein to use. and purified protein is too small + isoelectric point 8,5. one question: this antibody needs to be used for immunostaining as well as westernblotting. will the purified antibody work in all the application? Could you please send me the protocol. my email address is [email protected]

Biao Chen: I have cyanogen bromide-activated sepharose 4B so i will also try ph9.5 coupling buffer with it. thank you for your advice!

Hello Gurjot,

When I did the purification, I only tested on western blot. But the same protocol was previously used to purify other antibodies that were successfully used for regular western blot and immunofluorescence.

I'll contact you by e-mail!

Hi, You can make an affinity column wit your antigen, use the ready-to-use resin or column from Ge (Amersham/Pharmacia)  or Thermo (Pierce). Using acrilamide gel to purify your antibody from serum could be nice... but you only get minute amounts of your antibody.  

http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/17071601

Hi Liliana Marques,

I have a problem about purify antibody as Gurjot Kaur. I'm so excited when I found this topic. I want to purify antibody from serum, my protein is 60 kDa. Could you please send me your protocol "purify antibody by using polyacrylamide gel". My email address is "[email protected]" or "[email protected]". Thanks a lot.

Hi Liliana Marques,

I am facing trouble in purifying my antibody.I want to purify antibody from serum. I will highly appreciate if you can share your protocol'purify antibody by using PAGE. My email-id is [email protected].

It  would be a great help!!!

Thank you

 Hello Liliana Marques,

It would be great for me to try this protocol.

Please, could you send it to me? (mail. [email protected] or [email protected])

Thank you very much

Hi Liliana Marques,

I have a problem about purify antibody as Gurjot Kaur. I want to purify antibody from serum, my protein is 15 kDa. Could you please send me your protocol "purify antibody by using polyacrylamide gel"?

My email address is "[email protected]". Thanks a lot.

Hi Liliana Marques,

Could you please share your protocol for purification of antibody from serum?

email id- [email protected]

Hi Liliana Marques,

Could you please send me the protocol for purification of antibody from serum? I got the serum, did the western blot with serum directly (use serum and mix with blot buffer to incubate my membrane), but I did not see any thing on the membrane. I guess the concentration of my antibody is too low, so I need to purify it. My email is [email protected]