Abscisic Acid ELISA 试剂盒

• Assay plate
• Standard
• HRP-conjugate (100 x concentrate)
• Sample Diluent
• HRP-conjugate Diluent
• Wash Buffer (25 x concentrate)
• TMB Substrate
• Stop Solution
• Adhesive Strip

1. Prepare reagents, samples and standards as instructed.
2. Set a Blank well without any solution.
3. Add 50 µL standard or sample to each well.
4. Add 50 µL HRP-conjugate (1x) to each well (Not to Blank well).
5. Incubate 1 hour at 37 °C
6. Aspirate and wash 5 times.
7. Add 90 μL of TMB Substrate to each well. Incubate for 20 minutes at 37 °C. Protect from light.
8. Add 50 µL Stop Solution to each well. Read at 450 nm within 5 minutes.

Note: γ Kindly use graduated containers to prepare the reagent. γ Bring all reagents to room temperature (18-25 °C) before use for 30 min. γ Prepare fresh standard for each assay. Use within 4 hours and discard after use. γ Making serial dilution in the wells directly is not permitted. γ To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10 μL for once pipetting. γ Distilled water is recommended to be used to make the preparation for reagents. Contaminated water or container for reagent preparation will influence the detection result. γ Antibody (1x) - Centrifuge the vial before opening. Antibody requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of Antibody + 990 μL of Antibody Diluent. γ HRP-conjugate (1x) - Centrifuge the vial before opening. HRP- conjugate requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of HRP- conjugate + 990 μL of HRP- conjugate Diluent. γ Sample Extraction Buffer(1x)- If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Sample Extraction Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Sample Extraction Buffer(1x). γ Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Wash Buffer (1 x). 6 γ Standard Centrifuge the standard vial at 6000-10000rpm for 30s before opening. Dilute the Standard(10x) with Sample Diluent. A suggested 10-fold dilution is 50 μL of Standard(10x) + 450 μL of Sample Diluent. This diluted Standard (S7) serves as the high standard (10 μg/mL). Do not substitute other diluents. Mix the standard to ensure complete dilution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Pipette 250 μL of Sample Diluent into each tube (S0-S6). Use the diluted Standard (S7) solution to produce a 2-fold dilution series (below). Mix each tube thoroughly before the next transfer. Sample Diluent serves as the zero standard (0 μg/mL). Note:

• Kindly use graduated containers to prepare the reagent.
• Bring all reagents to room temperature (18-25 °C) before use for 30 min.
• Prepare fresh standard for each assay. Use within 4 hours and discard after use.
• Making serial dilution in the wells directly is not permitted.
• To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10 μL for once pipetting.
• Distilled water is recommended to be used to make the preparation for reagents. Contaminated water or container for reagent preparation will influence the detection result.
• Antibody (1x) - Centrifuge the vial before opening. Antibody requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of Antibody + 990 μL of Antibody Diluent.
• HRP-conjugate (1x) - Centrifuge the vial before opening. HRP- conjugate requires a 100-fold dilution. A suggested 100-fold dilution is 10 μL of HRP- conjugate + 990 μL of HRP- conjugate Diluent.
• Sample Extraction Buffer(1x)- If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Sample Extraction Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Sample Extraction Buffer(1x).
• Wash Buffer(1x)- If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved. Dilute 20 mL of Wash Buffer Concentrate (25 x) into deionized or distilled water to prepare 500 mL of Wash Buffer (1 x).
• Standard Centrifuge the standard vial at 6000-10000rpm for 30s before opening. Dilute the Standard(10x) with Sample Diluent. A suggested 10-fold dilution is 50 μL of Standard(10x) + 450 μL of Sample Diluent. This diluted Standard (S7) serves as the high standard (10 μg/mL). Do not substitute other diluents. Mix the standard to ensure complete dilution and allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. Pipette 250 μL of Sample Diluent into each tube (S0-S6). Use the diluted Standard (S7) solution to produce a 2-fold dilution series (below). Mix each tube thoroughly before the next transfer. Sample Diluent serves as the zero standard (0 μg/mL).

• It is recommended to use fresh samples without long storage, otherwise protein degradation and denaturationmay occur in these samples, leading to false results. Samples should therefore be stored for a short periodat 2 - 8 °C or aliquoted at -20 °C (≤1 month) or -80 °C (≤ 3 months). Repeated freeze-thawcycles should be avoided. Prior to assay, the frozen samples should be slowly thawed and centrifuged toremove precipitates.
• If the sample type is not specified in the instructions, a preliminary test is necessary to determinecompatibility with the kit.
• If a lysis buffer is used to prepare tissue homogenates or cell culture supernatant, there is a possibilityof causing a deviation due to the introduced chemical substance.The recommended dilution factor is for reference only.
• Please estimate the concentration of the samples before performing the test. If the values are not in therange of the standard curve, the optimal sample dilution for the particular experiment has to be determined.Samples should then be diluted with PBS (pH =7.0-7.2).