Firstly, I wanna purify one kind of antibody from serum which is specific to one kind of protein. Ex) protein XX. So, I coupled this protein XX to CNBr sepharose resin to make protein XX affinity column. I coupled 1 mg of this protein to 0.75 g CNBr resin, but when I checked the protein XX column's affinity, I found the capacity of this affinity column is so weak to bind with anti-protein XX antibody in serum. Then I wanna know what's the problem? If I coupled so small amount of protein to CNBr resin?
Proteins attach to CNBr Sepharose in random orientations, so one problem may that the antibody epitope(s) are partially obscured. Another problem could be that if the antibody has a structural epitope, damage to the protein structure during the coupling reaction may destroy the epitope. Next, the capacity of the column for antibody is limited because each antibody molecule (mol. wt. 150,000 for IgG) can only bind to one or two protein molecules. Lastly, the highest affinity antibodies are hard to elute from the column; only the lower affinity antibodies are recovered.
Proteins attach to CNBr Sepharose in random orientations, so one problem may that the antibody epitope(s) are partially obscured. Another problem could be that if the antibody has a structural epitope, damage to the protein structure during the coupling reaction may destroy the epitope. Next, the capacity of the column for antibody is limited because each antibody molecule (mol. wt. 150,000 for IgG) can only bind to one or two protein molecules. Lastly, the highest affinity antibodies are hard to elute from the column; only the lower affinity antibodies are recovered.
Hi YINGJI, can you elaborate on this statement about you wanting to "purify one kind of antibody from serum which is specific to one kind of protein"
Are you trying to isolate a specific antibody from polyclonal antisera?
I agree with Adam Shapiro on all accounts. If you are lucky there may be some antibodies against your protein that are specific to its denatured regions. We use 2mg of Ab to couple to one ml wet column volume of CNBr Sepharose (see attached protocol from GE). Each 1 gram dry powder gives 3.5 ml wet volume, so for 0.75 g, you'll have about 2.7-3 ml wet volume, which means you need to increase your protein by about six times of what you use. Good luck.
As stated above, please read the manual before doing the experiment! For CNBr (e.g. from GE) the binding capacity for an antigen is quite high and it is recommended to use 5-10mg of your protein per ml resin. Your 0.75g (>3ml) of resin would require at least 15mg of protein, so you're quite far from the optimum.
Thank for your all kind advices! I would try again, and I will increase the amount of protein.
Thank u so much,everyone~!
One thing to warn you is that the recommended amount of protein by GE is on the high side and can cause cross reactivity. So, do not go too high on protein amount to couple-stay within 2-5 mg/ml wet volume, no more.
We also would start with 5mg of protein per 1 ml of resin and most of the time we would get about 0.5 mg per 1.0 ml of resin capacity. Occasionally we would go as high as 10mg/ml and as low as 2-3 mg/ml of resin to address various binding issues.
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