I have an antibody against my protein of interest that I would like to use to help with purification. Unfortunately I haven't been able to find a lot of information about how to separate the protein from an antibody without damaging the protein itself. I need to purify this protein for further activity analysis. I've tried tagging it and following it up with elution from an HA column, but I haven't been able to successfully elute it from the HA column without using harsh conditions. This is an alternative I'm looking into. Any suggestions?
Thanks!
Hi Tami, there are many methods to dislodge proteins from antibodies. They mostly involve extreme pH or organic solvents.
If your capture antibody is a monoclonal, then you just have to find conditions (pH, organic solvents) that can release your target without denaturing it.
If your capture antibody is a polyclonal, then that makes things a bit more difficult.
If you are using a monoclonal antibody with a known linear epitope, you may be able to elute the captured protein using a peptide containing the epitope. It's expensive, but I have used this method successfully.
Also, have you considered conventional chromatography to purify your protein, such as ion exchange, gel filtration and hydrophobic interaction?
I'm not sure if it works in your case but there are cleavable spacers. Maybe you find some tag with such a spacer that is sensitive to a very defined protease which will not cut in your protein - and the remainings are not a problem for further steps.
Larry Haff (then Pharmacia) showed that isoelectric focussing works well with immobilised ampholines. We showed that electrophoretic desorption also works rather well. You need to hold down the antibody with CNBr activated Sepharose before you do this (see Anal Biochem 105, 1-5 1980).
Proteins differ in which denaturing agents will cause irreversible denaturation and which might not. Assuming that you have the antibody affixed to a column, if you use low pH to elute, then very quickly change the pH. A 'trick' is to collect what you elute off the column into tubes with strong buffer of proper pH to immediately change the pH. In addition to low pH, you can keep pH at a pH that your protein is stable in and add a chaotropic agent such as thiocyanate, then very quickly have a method ready to separate the protein from the thiocyanate. You will also want to quickly wash the antibody-resin with a buffer appropriate for antibodies so you can use it over again.
Thank you all for your suggestions! I think I'll try my hand at a few of these and leave an update on the outcome. Thanks again!
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