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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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immunoprecipitation protocol
Posted by: zuodan (IP Hidden, Unregistered user, )
Date: April 24, 2005 07:44PM
I want to know the protocol of immunoprecipitation in order to decide whether I can do in my lab.Thank you for your help.
Re: immunoprecipitation protocol
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 25, 2005 01:17PM
Hello,
I have used this approach to cross link the IgG to protein A, so you would not see it on the gel I have used the following IP procedure with great success 2.2.2.i Coupling anti-CrhC antibody to protein A immobilized on sepharose beads Coupling anti-CrhC to protein A was performed as described Harlow and Lane (1988). Briefly, 100 mg of lyophilized protein A immobilized on Sepharose CL-4B (Sigma) were swollen for 30 min at room temperature in 2 ml of immunoprecipitation coupling buffer (100 mM NaHCO3, 50mM NaCl, 1mM PMSF, pH 8.3). Anti-CrhC antibodies were mixed with swollen protein A beads at a final dilution of 1:50 and the mixture incubated at room temperature for 60 min with continuous end-over-end gentle rotation. The beads were washed twice with 10 volumes of borate buffer (0.2 M sodium borate, pH 9) followed by centrifugation at 1 000 g for 5 min. The beads were resuspended in 10 volumes of borate buffer, for crosslinking anti CrhC antibodies with protein A solid dimethyl pimelimidate dihydrochloride was added to a final concentration of 20 mM, and the mixture incubated for 30 min at room temperature. The reaction was stopped by washing the beads twice in 0.2 M ethanolamine pH 8, and then incubated for 2 hr at room temperature in 0.2 M ethanolamine with gentle mixing. After the incubation period, the 0.2 M ethanolamine was discarded after pelleting the beads by centrifugation for 1 minute at 14 000 rpm. The beads were resuspended in PBS with 1m M PMSF to be used for immunoprecipitation experiments.
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