I want to know what other possible tests you can do to make sure that the normal serum is removed in immunoprecipitation (the one added in preclearing). One test is with the lysis buffer on the preclearing steps. What other checks can be done? TIA
Easiest way is probably running the sample through a Protein A/G column to trap all IgGs and then checking by Western Blot if there are any (probably bovine) IgGs left. Dot Blot or ELISA like format might work, too.
The resin easily may be regenerated with an acidic buffer, e.g. 100mM glycine HCl, pH 2.5 in 0.9% NaCl.
Hello Debrah Almazan
Another method which you could use is to incubate the protein mixture with exactly the same components that will be used in the immunoprecipitation, except that a non-target, irrelevant antibody of the same antibody subclass as the IP antibody is used instead of the IP antibody itself.
This approach attempts to use as close to the exact IP conditions and components as the actual immunoprecipitation to remove any non-specific cell constituent without capturing the target protein.
Best.
It is difficult to identify low abundant proteins because of highly abundant human serum albumin (HSA). PureProteome albumin Magnetic Beads (MilliporeSigma, USA) can be used for the depletion of HSA from plasma following the manufacturer protocol. The PureProteome is the antibody conjugated bead specific for HSA that captures and removes albumin from samples. Dilute the Plasma 10fold with PBS and filter it through a 0.22 µm membrane. HSA depleted sample can be stored at -20°C.
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