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RESEARCH DIVISION Laboratory Manual

 
 

Immunoprecipitation from Eukaryotic Cell Extracts.

  1. Grow cells to subconfluence. Aspirate media and wash with ice cold PBS twice and aspirate all of PBS.
  2. Lyse cells with ice cold PLC-lysis with protease +/- phosphatase inhibitors (see Reagents). Use 1.0ml per 10cm plate, 3.0ml per 15cm plate. Add PLC buffer in cold room and place plates on rocker for 60'. Set refrigerated microfuge chilling down to 4°C. Swell 2.5mg protein A sepharose (P-3391, Sigma) per sample in PLC lysis buffer for 20' on wheel, spin down in benchtop and wash in PLC buffer twice to remove preservative. Resuspend protein A in PLC buffer at 50ul per sample.
  3. Scrap cells and collect lysate and add to tubes on ice. Spin at 13K for 10' at 4°C and collect s/n.
  4. OPTIONAL preclear with either 20ul normal rabbit serum (for a rabbit polyclonal IP) or 0.5ul anti-mouse Ig(for a mouse monoclonal IP) and 2mg protein A for 2hr at 4°C (worthwhile to reduce background). Add 5ul of antisera to lysate and 50ul protein A sepharose (see #2). The amount of lysate used depends entirely on how good the antisera is and the abundance of the protein. 1/5th to 1X 15cm plate per antisera is a reasonable range.
  5. Incubate on wheel at 4°C for 1hr to overnight. Spin out protein A sepharose in refrigerated microfuge (45 sec) and aspirate s/n to last 50ul. Add 1ml PLC lysis buffer and vortex well. Make sure sepharose beads are well resuspended.
  6. Spin out sepharose and repeat #5 for three-five washes. Remove all lysis buffer with a Gilson (through beads and tip flat on bottom to sieve out beads).
  7. Add 90ul SDS sample buffer and boil for 3'. Spin and load 90ul per lane on gel. Western transfer unlabelled lysates, Amplify (S35 labelled lysates) and dry and autoradiograph (I125, P32-labeled proteins).

NOTE:

  1. If lysate is labelled with S35 methionine washes must be performed in radioactive hood and microfuge.
  2. If the IP is run on a gel and western transferred to probe for the antigen, then be aware the secondary (anti-mouse, anti-rabbit etc) may react with the antisera present in the IP (heavy chain ca. 50Kd, light chain ca. 25Kd).
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998