Dissolve Protein or Carbohydrate in ELISA Coating Buffer at 1 µg/ml
Add CB to concentrated protein solution, (not protein to CB)

Coat 60µl at room temperature overnight or 2 hours at 37°C, in a humidified chamber
-60µl is minimum volume required to cover entire bottom of ELISA plate wells
-use same volume for all subsequent incubation steps
-do all incubations in humidified chamber
-Never allow wells to dry out

Wash plate 5 times with PBS-Tween by gently filling all wells with PBS-T from squirt bottle, then invert to empty

Block plate (optional) with PBS-T or PBS-T + 1% BSA for 30 minutes at room temperature

Incubate I° antibody (in PBS-Tween) at least 1 hour room temperature or 4°C overnight

Wash as before, 5 times

Incubate alkaline-phosphatase conjugated 2° (in PBS-Tween) antibody 1 hour room temperature

Wash 4 times with PBS-T, then once with TBS to wash away phosphate and Tween

Add freshly made PNPP substrate
1ml of ELISA substrate + 9ml of ELISA buffer (See next page)

Read on Microplate reader at 405nm, 1 minute intervals for approximately 15 minutes

(50mM Sodium Carbonate Buffer)

To 400ml Milli-Q water add:

Bring volume to 500ml with Milli-Q water

pH should be 9.80 to 9.95 Do not adjust pH.

10mg/ml anhydrous P-nitrophenyl phosphate

To 90 ml Milli-Q water add:

-1.42 g Sigma104 phosphatase substrate (has 6H2O)

-bring volume to 100 ml with Milli-Q water

-make 1 ml aliquots

-store at -20'C

To 80 ml Milli-Q water add:

-9.6 ml Diethanolamine

-Adjust pH to 9.8 with 1 to 2 ml Concentrated HCl

-Bring volume to 100ml with Milli-Q water

-Store at room temp in dark bottle

-Discard when solution is slightly yellow

using HCl and Diethanolamine