Bring all reagents and samples to room temperature for 20 minutes before use.
1. Wash Buffer
If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use.
Dilute 30ml (15ml for 48T) Concentrated Wash Buffer into 750ml (375ml for 48T) Wash Buffer with deionized or distilled water. Put unused solution back at 2-8°C.
1) Add 1 ml Sample Dilution Buffer into one Standard tube (labeled as zero tube), keep the tube at room temperature for 10 minutes and mix them thoroughly.
Note: If the standard tube concentration higher than the range of the kit,please dilute it and labeled as zero tube.
2) Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3ml of the Sample Dilution Buffer into each tube. Add 0.3ml of the above Standard solution (from zero tube) into 1st tube and mix them thoroughly. Transfer 0.3ml from 1st tube to 2nd tube and mix them thoroughly. Transfer 0.3ml from 2nd tube to 3rd tube and mix them thoroughly, and so on. Sample Dilution Buffer was used for the blank control.
Note: It is best to use Standard Solutions within 2 hours.
3. Preparation of Biotin-labeled Antibody Working Solution
Prepare it within 1 hour before experiment.
1) Calculate required total volume of the working solution: 0.05ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total volume.)
2) Dilute the Biotin-detection antibody with Antibody Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl Biotin-labeled antibody into 99μl Antibody Dilution Buffer.)
4. Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution
Prepare it within 30 minutes before experiment.
1) Calculate required total volume of the working solution: 0.1ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total volume.)
2) Dilute the SABC with SABC Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1μl of SABC into 99μl of SABC Dilution Buffer.)