A plate washer is recommended. However, washing may be done by gently squirting wash buffer into each well with a squirt bottle or by pipetting 300 l of wash buffer into each well and then flipping the liquid into a sink. After each wash, the plate should be thoroughly blotted on paper towels before turning upright. If using a plate washer, blotting is still recommended after the final wash. Add 200 l of TMB solution to each well with a multichannel pipette.
Wells are very full.
Add 50 l of stop solution with a multichannel pipette. Be sure all wells have turned yellow. If green color remains, continue mixing until green color turns to yellow. Wells are very full. Read in a plate reader at 450 nm.