Home | Jobs | News & Articles | Job Advice | Search | Protocols | Fun | @RealLabRat
Lab Rat with DNA

Post your resume
Candidates - post your resume

Search job listings
Candidates - search biotech jobs

Post job openings
Employers - post job openings

Email Login
New users
sign up!
HOME > Protocols > Cell Biology > Cell Culture Protocols > Protocol for Immunoprecipiation of Cultured Cells

Protocol for Immunoprecipiation of Cultured Cells

  1. Incubate cells at ~80% confluent in medium with 10% FBS lacking the labeled amino acid for approximately 1 hour at 37oC
  2. Remove media and replace with fresh media lacking the labeled amino acid with 10% FBS
  3. Add the radioactively labelled amino acid (approximately 100uCi/ml)
  4. Incubate at 37oC for 1hour to overnight. This step may require some optimization. While most proteins will be labeled after 1 hour, others may take overnight incubation
  5. Carefully remove radioactive media and dispose
  6. Add 6ml PBSTDS (4oC) to cells or cell pellet and incubate for 10 minutes
  7. Mechanically disrupt cells by repeatedly drawing mixture through a 21 gauge needle
  8. Wash incubation plate with an additional 4ml of PBSTDS (4oC)
  9. Centrifuge at 2,000rpm for 15 minutes in a refrigerated centrifuge
  10. Discard pellet
  11. Centrifuge supernatant at 40,000rpm for 20 minutes in a refrigerated centrifuge
  12. Discard pellet
  13. Aliquot approximately 1ml of supernatnat to 1.5ml eppendorf tubes
  14. Add approximately 1ug antibody to target protein and incubate for 1-2 hours at 4oC. The concentration requires optimization
  15. Add conjugate antibody. Several options are available here and I have commonly used agarose conjugates
  16. Incubate at 4oC on rocker platform for 12-24 hours (requires optimization - may need less time)
  17. Collect immunoprecipiatated protein by centrifugation at 2500rpm for 15 minutes at 4oC
  18. Wash pellet 3-4 times with 1ml PBSTDS
  19. Resuspend in 40ul gel running buffer
  20. Boil samples for 3 minutes to denature prior to SDS-PAGE analysis

Note: Please store, incubate, and dispose of radioactive materials responsibly!

theLabRat.com 2005. All Rights Reserved.