Anti-Cytochrome P450 1A1 + 1A2 antibody (ab4227)

  Immunoprecipitation - Cytochrome P450 1A1 + 1A2 antibody (ab4227)

ab4227 used in Immunoprecipitation.

The systems were cross-linked (X-L) with BS3, immunoprecipitated (IP) with ab4227, subjected to SDS-PAGE, and blotted with anti-CYP1A2.

Each of the reconstituted systems was cross-linked with the water-soluble, homobifunctional cross-linking agent BS3, which reacts with primary amino groups. Each 100µl reaction mixture was treated with 1 mM BS3 for 2 minutes at 4 °C and then quenched with 96µl of ice-cold 2radioimmune precipitation buffer and 500 mM glycine and placed in an ice bath. Samples remained on ice for 15 minutes before the addition of 4µl of ab4227. Samples were then allowed to incubate for 2 hours on ice. At the end of the 2-hour incubation, 100µl of 25% (w/v) Protein G-agarose bead slurry was added, and each sample was rocked/incubated for 1 hour at 4 °C. Samples were spun for 10 seconds at 3500g, the supernatants were transferred to clean microcentrifuge (1.5-ml) tubes, and the bead pellets were washed four times with 100µl of radioimmune precipitation buffer. After aspirating the final wash, 100µl of loading buffer was added to each sample. The samples were treated with 5µl of beta-mercaptoethanol before boiling for 10 minutes. 30µl of each sample was loaded onto 4–12% (w/v) SDS-polyacrylamide gels and run for 45 minutes at 30mA before running an additional 50 minutes at 200 V.The gels were transferred to nitrocellulose membranes and subjected to immune blot analysis.

Image from Reed JR et al, J Biol Chem. 2010 Mar 19;285(12):8942-52. Epub 2010 Jan 13. Fig 5.