Protocol (ELISA)

DAS ELISA Method

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

Incubate 4hrs at 37oC.

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 4oC.

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 37oC.

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.

REAGENTS: Coating buffer, Wash buffer, Extraction buffer, Substrate buffer

NB: The above protocol is used by us for the QC evaluations that are carried out by us on our reagents. Users have found that conditions and reagent volumes may be varied without substantially affecting the results obtained.

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 37oC.

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 4oC.

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 37oC.

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