- Proteins and Peptides
- Lysates and Cell Lines
How do I use the coat buffer?
Simply dilute the coat buffer 1:5 (for example, add 100mL 5x coat buffer to 400mL diH20 for a final volume of 500mL), add your protein antigen or antibody, let the solution stir for 15 minutes, and pipette onto the plate. As both coat buffers are concentrated 5x, crystalline precipitates may form in the bottle, especially when refrigerated. If this happens, gently warm the buffer until all crystals are dissolved. Do not let it boil.
How do I coat plates?
1. Dilute the 5x coat buffer by adding 1 part buffer to 4 parts deionized water (100mL coat buffer to 400 mL diH2O, yielding a total volume of 500mL) and mix for 15 minutes.
2. Dilute your antigen or antibody into the coating buffer (coating concentration varies significantly from less than 0.1ug/mL to over 10 ug/mL).
3. Let the solution stir (10 - 15 minutes) and pipette onto the plate (coating volume generally ranges between 50-300 uL per well).
4. Once added to the plate, incubate the coating solution from 3 - 24 hours at room temperature (RT) protected from light (minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a humidified storage box, and cover).
5. After incubation, dump or aspirate the coating solution out of the wells.
6. Wash the plate 2 - 4 times with ICT’s wash buffer.
7. Aspirate and pipette one of ICT’s block buffers onto the plate at a higher volume than the coating solution (300-400uL per well).
8. Once added to the plate, incubate the block buffer from 3 - 24 hours at RT protected from light (minimize evaporation by individually covering each plate with a plate sealer, wrapping a stack in plastic wrap, or placing plates in a humidified storage box, and cover).
9. Aspirate the block buffer.
10. The assay can be run at this point, or the plate can be dried and packaged for later use.
11. Dry the plate by letting it sit on the bench top from 2 - 24 hours (but protected from light - loosely cover with aluminum foil), or dry in a drying oven from 2 - 24 hours at RT or warmer.
12. When dry, seal the plate in an air-tight foil pouch with a desiccant and store at RT or 2°-8°C protected from light.