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 > ELISA > Interferon-Gamma ELISA Protocol

Interferon-Gamma ELISA Protocol

  1. Coat plate with 50ul of a 2.0ug/ml of monoclonal rat anti-murine interferon gamma antibody in 50mM sodium bicarobonate buffer (pH 9.4)
  2. Incubate plates 24 hours at 4oC
  3. Aspirate wells
  4. Wash five times with PBST
  5. Add 200ul of 5% milk in PBS (Blotto blcok)
  6. Incubate 60 minutes at room temperature
  7. Wash five times with PBST
  8. Make dilutions from 2000 - 0 pg/ml of mouse interferon-gamma in 4% FBS in PBS
  9. Add samples to be tested. If necessary, dilute samples as in step 8
  10. Incubate 1 hour at room temperature
  11. Add 50ul of 0.1ug/ml biotinylated anti-mouse interferon-gamma antibody in 4% BSA in PBS
  12. Incubate 1 hour at room temperature
  13. Wash five times with PBST
  14. Add 100ul of streptavidin labele horseradish peroxidase in 4% BSA in PBS
  15. Incubate 30 minutes at room temperature
  16. Wash five times with PBST
  17. Add 100ul of TMB substrate with H2O2 to each well
  18. Incubate 20 minutes at room temperature in the dark
  19. Add 50ul stop solution (1M H2SO4)
  20. Read absorbance at 450nm
  21. Read absorbance at 570nm (value at 570nm is subtracted from 450nm value to control for optical abnormalities in the ELISA plates)

Note: Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.

theLabRat.com 2005. All Rights Reserved.