I am planning an immunoprecipitation experiment using Mouse monoclonal [11C9] to Mannan Binding Lectin/MBL (ab26277) antibody to immunoprecipitate for MBL2 in human serum. There are many protocols online for immunoprecipitation utilising cell cultures, where the recommended amount of cells tends to be 10^6 to 10^7, however not so many that utilise human serum.
Reading online, the optimal total protein load of immunoprecipitation seems to be 1-5 mg/mL, with 0.1 mg/mL being the minimum recommended load. Considering that the normal range for total protein in human serum is 60-83g/L (average: 71.5 g/L), loading 5 mg of total protein for immunoprecipitation would mean I need 69.9 mL. Alternatively, loading the minimum (0.1 mg), I would need 1.395 mL of human serum based on my calculations.
I cannot afford to be using 1.395 mL of human serum in my experiment due to lack of sample volume. I was wondering if anyone can share the amount of human serum they've used for immunoprecipitation before, where it was successful. Thank you in advance.
I think you want to IP a protein from cell culture. You should titer your serum in a simple capture format ELISA. Be sure to wash the cells with PBS and then harvest their supernatant with no media. Then you can plate the sup directly to run the ELISA.
John Carter Thank you for your response.
The aim of the experiment is to perform western blotting to detect a different protein of interest in the precipitated MBL2 human serum, and so I am not sure if ELISA is appropriate for this... Is there any other ways to go about this?
You're correct that Ab often perform quite differently in Western versus ELISA. But it's something you can try.
What makes you think your antiserum works at all? if you have any titer measurement you can use that to estimate a working dilution for IP.
Or you could always just guess. A dilution of 1:1000 should work, if the antiserum is reasonably potent. You may be able to go down to 1/10k or 1/100k.
Follow your local rules for use of human tissue in lab research. I recommend you always heat treat human serum to kill viruses, and then still treat the reagent as potentially infectious. My niece caught HepB from handling a human serum sample is a hospital lab.
- Badges
- Science topic