Antibody Purification

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achaud
achaud's picture
Antibody Purification

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.

Sandy
Sandy's picture
achaud wrote:I want to purify

achaud wrote:

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

samm
samm's picture
The best way to concentrate

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)

Paul Weinreb
Paul Weinreb's picture
My suggestion - just load the

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.

MadScientist74
MadScientist74's picture
pw_18 wrote:My suggestion -

pw_18 wrote:

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.

samm
samm's picture
Keep in mind that that

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.

bwbrian
bwbrian's picture
Different antibodies have

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

Backialakshmi D...
Backialakshmi Dharmalingam's picture
Hi Achaud,

Hi Achaud,

The following protocol is the one which i am using to purify IgG followed by dialysis. It gave me a good result.

Necessary buffers:
1. Glycine buffer (0.1 M) pH 9.0 - For washing abd binding

2. Glycine buffer (0.1M) pH 2.7 for eluting the samples.

Tris HCL : 1M pH 8.5 (should be added after eluting the samples from the column to bring down the pH)

Dialysis buffer 1X PBS
You can prepare stock 10X PBS

Procedure in short:

Keep in mind all the buffers, samples that flows thru the column must be filtered before use . I use 0.2µm syringe filters. Important to note down is keep the flow rate between 3 to 4 so that it makes a great

Fix the column in the stand. Allow 5 to 10 CV  (may be 5 to 10ml) of Glycine buffer of pH 9.0 to equilibrate the column.

Run the sample twice in the column to be sure about the binding. (I even store the samples in Refrigerator till my result comes).

Now again run the column with 10 CV of glycine buffer with pH 9.0. Once this step is done, add 10ml of glycine buffer of pH 2.7for eluting the sample.

Simultaneously collect 500µl of sample in 10 different tubes. After eluting them add 50µl of 1M Tris HCL to each tubes  to bring down the pH from acidic condition.

Be sure that the column doesnt have any air stacks or bubbles as this may have  a big impact on your column. The columns are really quite expensive.

After the elution of samples please dont forget to re-equilibrate the column with glycine buffer pH 9.0.

For better results, read all the tubes in spec to find out the concentration of the protein present in each tubes. Then select the samples according to the concentration of protein. (Always used to select the samples which has concentration greater than 1.8)

Now pool all those samples which has higher concentrations of proteins and pour them in to the dialysis bag. (preparing the dialysis bag for the process is also important).

Leave the dialysis bags in a big tank which has 1X PBS for half an hour before you send the samples in to the bag.

Pour the sample in to the bag and leave it in the 1X PBS and keep in the refrigerator mixer in slow mixing level. 

Change the 1X PBS in the tank at frequent time intervals (lets say for each five hours u can change the 1X buffer in the tank) for 36 hours. Then you can read the IgG concentration of the sample.

Note:

If you are not able to get good yield in this process you can try ÄKTA FPLC in which you can use either  HisTrapp_FF_Crude_1ml

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for smaller volume of samples or you have different 5ml columns which are pre- packed are free to pack by yourself using Sepharose or Sephadex etc. The use of matrix differs with the amount of sample you have in your hand.

Good Luck

Paul Weinreb
Paul Weinreb's picture
BAVE - I think achaud has

BAVE - I think achaud has probably completed graduate school in the 5 1/2 years since this was originally posted, so this protocol may not be of much help.   ;)

But perhaps now he/she has his/her own graduate students who need help with antibody purification...