A simple ”quick and dirty“ method: ammonium sulfate precipitation followed by electrophoresis to purify the protein.

right, good idea, but is it possible to do that in denaturing conditions? Ag and Ab are still in complex in the sample. Abs will precipitate with Ag bound, won't they?

Why not? It's a very old method to purify antibodies (or proteins) first published in 1942 (J Gen Physiol. 1942 Jan 20;25(3):465-85.

PURIFICATION AND CRYSTALLIZATION OF DIPHTHERIA ANTITOXIN.

Northrop JH.)

sure it's great simply and useful method, but If I do it in native state, immunoglobulins will precipitate with bound antigens; and on the other hand If I use denaturing conditions, it will alter a solubility of globulins and I will precipitate something else. I need to separate Ab and Ag and isolate only antigen (many salmonella proteins) from IgGs. that's a quite problematic

You can bind antibodies and Ab-Ag-complexes on an protein-A (particle, sepharose, ...). Use a gradiend elution procedure. Start with PBS and increase the KCSN-concentration up to 3 mol/L. Antibodies allone will elute at another point than antibdies with bound antigen and the antigen itself.

I would go for the Protein G. It is relatively fast .

Pierce offers several variations on biotinylation kits for antibodies. Once biotinylated you could capture the Ab-target complex on a streptavidin column (also sold by pierce) and then elute only the captured target since the streptavidin biotin bond is so strong. You can do this on a magnetic bead scale or as a larger column depending on your needs.

Roald Nezlin- Weizmann Inst. of Sci., Israel.

The simplest way is column gelfiltration in acid or other separating buffers - most probably IgG Ab will be separated from bacterial proteins.

ok, let's go to the basics: the interaction between the antibody and their respective antigen is a protein-protein interaction featuring hidrogen bonds, ionic bonds, hydrophobic interaction, and so on. I remember a research, very old one, where they had the same problem, elute the protein from the antibody previously conjugated to the column (however, native conditions). They used ionic strength (increasing salt concentration, in this case up to 2M MgCl2). It works like a ionic exchange column. I guess that even if you had your sample in denaturing conditions, you could try this, to my knowledge, the simplest way to obtain your salmonella protein. Let us know how did it go, good luck!

so, I've done ammonium sulphate precipitaion, in native state and after adding some glycine. without glycine (only Ab-Ag in PBS), IgG precipitated with Ag bound (according to 1D gel electrophoresis). More glycine added resulted in better resolution of salmo proteins on gel, but incomplete precipitation of antibodies (less amount than before, but still abundant)

I had done column separation before, in the column there was a protein G for binding Ig, so it's protein-protein interaction as well. The problem is that in lysate, there is many salmo proteins with different properities, some of them may be simillar to immunoglobulis (pI, MW), so it's difficult to separate only IgG from all other proteins. (and I need them all:))

I believe in some other method - to bind antibodies covalently to some particles (protein A/G crosslinked, or MHS-activated magnetic beads), and after elution I hope I'll get only salmo proteins without antibody contamination

thanks a lot for all your suggestions and comments

There are more than a hundred of proteins, so I want to put the sample onto 2D gel. It's complicated sample for 2D because of abundant antibodies, that's the reason I wanted to separate them. Thank you for reply, I'm gonna look through the forum you mentioned.

Hi,

we are offering covalent binding of proteins to our system. This are Polystyrene Beads with closed surface. Please check here: http://pluriselect.com/customized-products.html after elution e.g. denaturing or pH or other the antibodie will stay on the plead and does not interfere with your following analysis. In case you have questions please contact us at any time.

If the size of the Salmonella protein is ~20kd different in size than the ~150kd porcine IgG, then I would equilibrate a size exclusion column (e.g. 1x 25-30cm Sephadex G200 column) with 0.1M citrate (pH 2.9, with or without 0.15M NaCl- I'd go without to begin) and pass (~1 ml, don't overload) the sample over the column. At that pH, the antibody and the Salmonella protein will disassociate and separate from one another based on size. We routinely elute our antibodies (polyclonal, monoclonal, scfv) using citrate. Then adjust the pH to ~ neutral with NaOH. The antibodies seem to do quite well under those conditions. I'm not sure how your protein wil fair though. What's nice about taking such an approach is that it's simple, just requires a size exclusion resin and a column. The resins are quite stable and just need to be washed with 10-20 column volumes of buffer to clean them. A chromatography system is not needed as the column can be run using gravity. If you're not using a chromatography system, the downside will be that you will need to collect the eluted column fractions by hand, unless you have a fraction collector.

Thanks a lot for all your answers. I appreciated your attention and time spent in order to help me with the protocol.

With most of the 16 answers clear and do-able, my suggestion is to reduce the pH of your immune complex suspesion down to say 3.0 which will split the noncovalent bonds between antigen&antibody; lowering ionic strength can do the rest. This can be followed by ultracentrifugational separation releasing two peaks each one susceptible for preparative enrichment of the now separated compounds; this answer goes along with the one by Dr. Khosravi.

In dissociation IC Elisa assay, we employed, for several biological fluids, dilute sample 1:100 (1 µl + 100 µl) with dissociation buffer (PBS buffer with 1.5% BSA and 0.2 M glycine-acetate), pH 2.5 and/or pH 3.5 to a 500 µl final volume and incubated for 20 min. at room temperature. The treated diluted sample is then pipetted into the sample reservoir of Microcon centrifugal filter device, YM-10 (10,000 mw cut-off by Millipore) and centrifuged at 8,000 × g for 20 min. at RT. The sample reservoir is then separated from the flow through, placed inverted into a second tube and centrifuged at 1000 × g for 3 min. at RT. The collected solution, containing the antibody dissociated from the antigen, is adjusted to pH 7.0 with 1 M Tris buffer, pH 9.0. The retentate volume is reconstituted to the initial volume (500 μl) with ELISA dilution buffer (PBS with 1.5% BSA and 0.1% Tween-20) and then dispensed in ELISA MTP at several dilutions to determine the antibody titer.