ELISA Protocol to Detect Protein Interactions – Enzyme-Linked Immunosorbent Assay

ingredient
Nunc microtiter plates (binding media) 475 094 F96 polysorp (RIA plates)
Maxisorp F96 442 404

ABTS (Available in Sigma, for example)

buffer A
1M disodium hydrogen orthophosphate (20ml water)
1M citric acid (16mL water)
Combining and dilute 164 ml, pH to 4.0
Store in the dark at room temperature

Note:

  • Do the samples in triplicate
  • + 60uL of each solution at the time of each well; wash / rinse applied to fill the well and removed by inverting the plate and throw out the excess solution
  • Note that all proteins (including BSA) using 1x PBS as diluent
  • Variations include applying a particular protein or 10x using serial dilutions
  • Can use a multichannel pipette to accelerate the delivery of solutions for microtiter plates

Procedure

  • Plate Protein 1 overnight at 37 degrees in the incubator (cover with pipette tip box 1mL) – dilute protein to 1 up to 100 ug / mL
  • Rinse with PBS
  • Block with 4% BSA in PBS, 1 hour
  • Rinse with PBS
  • Add Protein 2 + 0.1% BSA
  • Incubate 1-4 hours at room temperature or at 37 degrees Celsius
  • Wash 3x with PBS-Tween
  • Incubate 1 hour at room temperature with 3 Protein + 0.1% BSA
  • Wash 3x with PBS-Tween
  • Incubate 1 hour at room temperature with 4 protein or antibody, the conjugate – a peroxidase (dilute peroxidase-conjugated protein or antibody 1/1000 with PBS-Tween) + 0.1% BSA
  • Wash 5-6x with PBS-Tween
  • developing a solution (5 mg in 10 mL ABTS Buffer A + 10ul H2O2) made just prior to use and kept in the dark before use
  • Wait 15 minutes (put foil around it to prevent light from reaching it)
  • Read microtiter plate with a plate reader at 405nm

To control, remove any proteins or BSA.
Other controls can be a specific primary antibody to replace the usual one, and replacing BSA Protein 1.