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.