I would like to purify IgG from mouse serum. I have tried melon gel purification according to the manufacturer's recommendation, but after SDS page I realized I had a lot of contaminants (even when I performed ammonium sulfate purification). I have followed the protocol and tried to apply recommendations from the troubleshooting section and I still cannot purify IgGs with melon gel. I have read that mouse transferrin may interact with the gel, but I know other labs have been able to use this method.
Then I tried to use the NAb Protein A/G Spin Kit, 0.2mL from Thermofisher. This time I was able to purify IgGs without contaminants but my yield is very low (around 0.3mg/ml and mouse serum should contain 2-5mg/ml of IgG). I would like to increase this yield without having to use more serum as I am limited in quantity (I can do 200µl input max).
If you have already purified mouse IgG from serum, I would be grateful to learn your tips and the method you used
Thank you very much
You could try a simple caprylic acid precipitation of non-immunoglobulin proteins of a normal mouse serum (first, for precaution) and verify if it suits your needs/expectations of purity, before running your actual valuable sera. Caprylic acid is added to the serum to achieve a final 5% vol/vol concentration, and then it is stirred at medium to vigorous speed for 30 min at RT. The albumin precipitate is separated by centrifugation, and the supernatant (containing mainly IgG) can be collected and subjected to membrane ultrafiltration (12 kDa pore) using a small centrifugal device, to get rid of caprylic acid in 2-3 washed with your preferred buffer. You can check an electrophoretic image of the outcome in page 35, fig.3.4 of the manual downloadable here in researchgate:
Book Manual de Metodos Inmunologicos (v05.1)
hope this may help
Antibody purification involves isolation of antibody from serum (polyclonal antibody), ascites fluid, or from the culture supernatant of a hybridoma cell line (monoclonal antibody). Purification methods range from very crude to highly specific. The level of purification necessary to obtain usable antibody depends upon the intended application(s) for the antibody. Antibodies may be purified by class (e.g., IgG) without regard to antigen specificity, or affinity purified to an immobilized antigen that is antibody specific.
Protein A/G is a genetically engineered protein with combined IgG binding profiles of both Protein A and G. It contains four Fc binding domains from Protein A and two from Protein G, providing additive class-specific binding properties for Protein A and G.
