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

Biochemistry, Molecular Biology, and Cell Biology Protocols >> ELISA (Enzyme-Linked Immunosorbent Assay) Protocol to Detect Protein Interactions

Materials
Nunc microtiter plates (medium binding) polysorp F96 475094 (RIA plates)
Maxisorp F96 442404

ABTS (Available at Sigma, for example)

Buffer A
Disodium hydrogen orthophosphate 1M (20mL water)
Citric Acid 1M (16mL water)
Combine and dilute to 164 mL, pH to 4.0
Store in the dark at room temperature

Notes:

- Do samples in triplicate
- +60uL of each solution to the well each time; washes/rinses are applied to fill the well and removed by inverting the plate and flinging out excess solution
- Note that all proteins (including BSA) use 1x PBS as the diluent
- Variations include applying certain proteins using a serial or 10x dilution
- Can use multichannel pipet to speed up delivery of solutions to microtiter plates

Procedure

- Plate Protein 1 overnight at 37 degrees in incubator (cover it with a 1mL pipet tip box) - dilute protein to 1 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 Protein 3 + 0.1% BSA
- Wash 3x with PBS-Tween
- Incubate 1 hour with room temperature with Protein 4 or antibody, which is conjugated to - peroxidase (dilute the peroxidase-conjugated protein or antibody 1/1000 with PBS-Tween) + 0.1% BSA
- Wash 5-6x with PBS-Tween
- developing solution (5mg ABTS in 10mL of Buffer A + 10uL H₂O₂) – make just before use and keep in the dark prior to use
- Wait 15 minutes (put foil around it to prevent light from reaching it)
- Read the microtiter plate with a plate reader at 405nm

For controls, omit each protein or BSA.
Another control could be a nonspecific primary antibody replacing the regular one, and BSA replacing Protein 1.