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Bring all kit components and samples to room temperature (18-25°C) before use. Dispense 10 µL of lysis buffer solution into 100 µL specimens, mix and stand for one hour (The proportion of lysis buffer and specimens shall be no less than 1:10). (NOTE: This step is required when the sample is cell culture fluid & body fluid & tissue homogenate, If the sample is serum or blood plasma, then this step should be skipped.) Wash Solution Dilute 10 mL of Wash Solution concentrate (100X) with 990 mL of de-ionized or distilled water to prepare 1,000 mL of Wash Solution (1X).
It is recommended that all Calibrators and Samples be added in duplicate to the Microtiter Plate.1. Secure the desired number of coated wells in the holder then add 100 µL of Calibrators or Samples to the appropriate well of the antibody pre-coated Microtiter Plate.2. Add 50 µL of Conjugate to each well. Mix well. Mixing well in this step is important. Cover and incubate for 1 hour at 37°C.3. Wash the Microtiter Plate using one of the specified methods indicated below:4. Manual Washing: Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Fill in each well completely with diluted wash solution, and then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure five times for a total of five washes. After washing, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame. Complete removal of liquid at each step is essential to good performance.5. Automated Washing: Wash plate five times with diluted wash solution (350-400 µL/well/wash) using an auto washer. After washing, dry the plate as above. It is recommended that the washer be set for a soaking time of 10 seconds and shaking time of 5 seconds between each wash.6. Add 50 µL Substrate A and 50 µL Substrate B to each well, subsequently. Cover and incubate for 10 minutes at 20-25°C. (Avoid sunlight).7. Add 50 µL of stop solution to each well. Mix well.8. Read the optical density (O.D.) at 450 nm using a microtiter plate reader immediately.