Step 4. For the Buffer Only background controls, add 20 L of Complete Reaction Buffer to the appropriate wells. Each well of the assay should now have 20 L volume.
Step 1. Cover the plate and protect from light. Incubate at room temperature for 3060 minutes.
Note: You may need to optimize reaction time for your individual needs.
C: ADD BINDING SOLUTION AND READ FP
Progressive Binding Solutions preparation
Prepare the Progressive Binding Solutions according to the plate diagram in Table 5
Note: For Binding Solution optimization information, please refer to the IMAP Progressive Binding System Application Note. Binding Solutions are listed as % Progressive Buffer A/% Progressive Buffer B/dilution of Progressive BR.
Step 1. Prepare 15 mL of 100/0/1:400 Binding Solution by adding 37.5 mL BR to 15 mL of 1X Buffer A.
Step 2. Prepare 15 mL of 75/25/1:600 Binding Solution by adding 25 mL BR to 11.25 mL of 1X Buffer A + 3.75 mL of 1X Buffer B.
Step 3. Prepare 10 mL of 60/40/1:1200 Binding Solution.
a. Mix 6 mL of 1X Buffer A with 4 mL 1X Buffer B to make Binding Buffer.
b. Add 30 L of BR to 270 L of 0.1 N HCl
and mix. This is an intermediate 1:10
dilution of the BR into 0.1 N HCl. Save