Reagent Preparation
Bring all reagents and samples to room temperature for 20 minutes before use.
Wash Buffer
Standards
Preparation of Biotin-labeled Antibody Working Solution
Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution
Assay Procedure
1.Add the Standard working solution to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100 μL for each well).Cover the plate with the sealer provided in the kit. Incubate for 90 min at 37 °C.
2.Remove the liquid out of each well, do not wash. Immediately add 100 μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37 °C.
3.Aspirate or decant the solution from each well, add 350 μL of wash buffer to each well. Soak for 1~2 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times.
4.Add 100 μL of HRP Conjugate working solution to each well. Incubate for 30 min at 37 °C.
5.Aspirate or decant the solution from each well, repeat the wash process for five times as conducted in step 3.
6.Add 90 μL of Substrate Reagent to each well. Cover with a new plate sealer. Incubate for about 15 min at 37 °C. Protect the plate from light.
7.Add 50 μL of Stop Solution to each well.
8.Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.
Assay Precision
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CXCR4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CXCR4 were tested on 3 different plates, 20 replicates in each plate.
Both intra-CV and inter-CV are < 10 %.
Storage Comment
An unopened kit can be stored at 2-8°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
Note
An unopened kit can be stored at 2-8°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
Restrictions
For Research Use only