To coincide with the introduction of our new Protein G antibody purification resin, we thought that we’d do a quick introduction to go over the steps and considerations you may need for antibody purification. Purified antibodies can be a powerful research tool, but the steps necessary to purify them correctly and efficiently can be pretty tricky to nail down without a good amount of knowledge, and some hard work. Hopefully these recommendations will help set you on your way to getting that purified antibody you’ve been waiting for.
The first step in making sure your purification is on the right track is to make sure that you’re using the correct purification method. Depending on your final use, you may not need a highly purified antibody, and may be able to just use the tissue culture supernatant or antiserum if your assay isn’t concentration dependent. If you do require a more purified antibody, then an affinity purification is one of the fastest and easiest ways to filter the total immunoglobulin amounts out of different lysates or media.
In order to get the best yield from your affinity purification, it’s important to match the class of your desired antibody to the purification resin you plan to use. For example, if the antibody you’re attempting to purify is a monoclonal Rat IgG2a you would want to use a Protein G resin, due to its much higher ability to bind that subclass of IgG than Protein A. Alternatively, if you’re attempting to purify a polyclonal Rabbit antibody, you might want to choose a Protein A resin due to its slightly higher binding ability. If you are unsure about which resin would be more effective for your needs, most manufacturers will sell a test kit of their different resins in order for you to test them out for yourself.
Once you have the correct resin you want to use, it’s important to carefully go over the purification process. Depending on the sample you are trying to purify, it may need to be filtered or centrifuged to remove any excess particulate matter that may decrease binding ability or clot the column during purification. A correct pH is also essential for the antibody to bind to the Protein A/G resin, so it’s very important that the sample is pH’d before adding to the column. Typically for binding , a pH of 8.0 is used, but some research shows that certain subclasses bind better at a lower pH. Elution conditions are also very pH dependent, as the antibody will only dissociate from the Protein A/G if the pH is very low, typically 2.8. As the antibodies will be pretty unstable at such a low pH, after purification you will need to either preform a buffer exchange or neutralize the eluate with 1M Tris-HCl (pH 8.5).
Hopefully these steps will help get your research on the right track. There are a lot of great resources out there to help you continue your research with your purified antibody, and soon you’ll be able to harness all the ability that purified antibodies can offer. Please contact us with any questions at firstname.lastname@example.org.
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