I want to purify my IgG antibody and fear that dialysis will give an unreasonably low yield. More specifically, I want to gid rid of the all the ascites. Any suggestions as to how I should proceed?
Thanks.

achaud said:

I want to purify my IgG antibody and fear that dialysis will give an unreasonably low yield. More specifically, I want to gid rid of the all the ascites. Any suggestions as to how I should proceed? Thanks.

When you are dealing with large quantities of ascites it is always recommended to get your IgG fraction by ammonium sulphate precipitation followed by dialysis . Because of the high concentration of the antibody in ascites fluids you will recover a large amount. Other choices are to use affinity columns, they could be expensive and you might need some purifications before loading the column. Call the following companies for advise:
1- BioRAd, the maker of Affi-Gel column at:800-424-6723
2-Millipore corporation at: 800-426-4266

The best way to concentrate ascites fluid is by a 2 step (~42% final) ammonium sulfate cut. You can then dialyze out the salt using one of the small mini-dialysis systems now available. (alternately, use a 1.5ml eppendorf, punch out the roundel in the lid, without damaging the lid or fastener, insert a square of dialysis membrane, mount on a glass rod, and dialyse in a 400 ml beaker)

My suggestion - just load the whole mess on a protein A affinity column and be done with it. You may need to dilute the ascites first to reduce the viscosity, and you'll spend some money on the protein A column but its certainly the easiest way to get what you need.

pw_18 said:

My suggestion - just load the whole mess on a protein A affinity column and be done with it. You may need to dilute the ascites first to reduce the viscosity, and you'll spend some money on the protein A column but its certainly the easiest way to get what you need.

This is probably the easiest and best way... especially if you are doing IPs later or for WB.

Keep in mind that that different species Abs have different affinities to Prtn A or G - some hamster or even mouse Abs just don't bind to Prtn A. Rabbit Abs are just fine, so if you have those, Prtn A-sepharose might be the easiest way to go.

Different antibodies have variable binding to Protein A and Protein G. There are at least two other options out there: Protein A/G and Protein L.

Protein A/G is a fusion protein that combines the binding sites, and thus the specificities, of Protein A and Protein G. This makes the choice of Protein A vs Protein G a non-issue.

Protein L is completely different. It's another bacterial protein that has great specificity for antibodies, but instead of binding to the heavy chains, it binds light chains only. Specifically it only binds certain kappa light chains, mouse kappa1 is the most common mouse light chain and it binds well to Protein L. It obviously won't work for everything, but it really stands out for some antibody purifications. For example, single chain variable fragments (ScFv) can be purified with Protein L, but not Protein A or G. Another example is monoclonals from culture supernatants that have been supplemented with FBS. Since bovine antibodies don't bind to Protein L, the monoclonals can be recovered in remarkably pure form.

Check out this free handbook from Pierce for more information:
http://www.piercenet.com/Objects/View.cfm?Type=Page&ID=61E3C646-3EEA-47DD-A9AF-1DB253C7EAB6