Buffer Recipes

DAS-ELISA Method

Prepare coating antibody (MAb) in coating buffer at recommended dilution and dispense (200 µL) into wells of an ELISA plate.

Incubate 4hrs at 37^oC.

Wash plate x4 with wash buffer and blot dry.

Prepare sample by homogenising plant material in extraction buffer 1/10 (w/v) and dispense (200 µL) into test wells. Incubate overnight at 4^oC.

Wash plate x4 with wash buffer and blot dry.

Dilute antibody-AP conjugate at recommended dilution in extraction buffer and dispense (200 µL) into test wells. Incubate 2hrs 37^oC.

Wash plate x4 with wash buffer and blot dry.

Prepare substrate 1mg/mL in substrate buffer and dispense (200 µL) into test wells. Incubate at ambient for 1-2hrs.

Read OD of test wells at 405n.

Buffer Recipes

Coating buffer substance         Amount
Na₂CO₃                                         0.8g
NaHCO₃                                        1.4g
1% NaN₃                                       10mL
H₂O                                               500mL
pH 9.6

Wash buffer
Phosphate Buffered Saline + 0.05% Tween 20

Extraction buffer substance     Amount
Polyvinyl pyrrolidone                      10g
Bovine serum albumin                    1g
1% NaN₃                                        10mL
Wash buffer                                   500mL
pH 7.4

Substrate buffer substance      Amount
Diethanolamine                             48.5mL
1% Sodium azide                           10mL
H₂O                                               500mL
pH 9.8

1mg p-nitrophenyl phosphate per mL substate buffer   

Printable version of ELISA Buffer Recipes