Mouse E Cad E Cadherin Elisa Kit Research Use Only Sandwich Method

Mouse E-Cadherin,E-Cad ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The Microelisa stripplate provided in this kit has been pre-coated with an antibody specific to E-Cad. Standards or samples are added to the appropriate Microelisa stripplate wells and combined to the specific antibody. Then a Horseradish Peroxidase (HRP)- conjugated antibody specific for E-Cad is added to each Microelisa stripplate well and incubated. Free components are washed away. The TMB substrate solution is added to each well. Only those wells that contain E-Cad and HRP conjugated E-Cad antibody will appear blue in color and then turn yellow after the addition of the stop solution. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to the concentration of E-Cad. You can calculate the concentration of E-Cad in the samples by comparing the OD of the samples to the standard curve.

Materials provided with the kit

Procedure

Dilution of Standards

1.Ten wells are set for standards in a Microelisa stripplate. In Well 1 and Well 2, 100μl Standard solution and 50μl Standard Dilution buffer are added and mixed well. In Well 3 and Well 4, 100μl solution from Well 1 and Well 2 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. 50μl solution is discarded from Well 3 and Well 4. In Well 5 and Well 6, 50μl solution from Well 3 and Well 4 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. In Well 7 and Well 8, 50μl solution from Well 5 and Well 6 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. In Well 9 and Well 10, 50μl solution from Well 7 and Well 8 are added respectively. Then 50μl Standard Dilution buffer are added and mixed well. 50μl solution is discarded from Well 9 and Well 10. After dilution, the total volume in all the wells are 50μl and the concentrations are 24ng/ml,16 ng/ml,8 ng/ml,4ng/ml and 2ng/ml, respectively.

2. In the Microelisa stripplate, leave a well empty as blank control. In sample wells, 40μl Sample dilution buffer and 10μl sample are added (dilution factor is 5). Samples should be loaded onto the bottom without touching the well wall. Mix well with gentle shaking.

3. Incubation: incubate 30 min at 37℃ after sealed with Closure plate membrane.

4. Dilution: dilute the concentrated washing buffer with distilled water (30 times for 96T and 20 times for 48T).

5. Washing: carefully peel off Closure plate membrane, aspirate and refill with the wash solution. Discard the wash solution after resting for 30 seconds. Repeat the washing procedure for 5 times.

6. Add 50 μl HRP-Conjugate reagent to each well except the blank control well.

7. Incubation as described in Step 3.

8. Washing as described in Step 5.

9. Coloring: Add 50 μl Chromogen Solution A and 50 μl Chromogen Solution B to each well, mix with gently shaking and incubate at 37℃ for 15 minutes. Please avoid light during coloring.

10. Termination: add 50 μl stop solution to each well to terminate the reaction. The color in the well should change from blue to yellow.

11. Read absorbance O.D. at 450nm using a Microtiter Plate Reader. The OD value of the blank control well is set as zero. Assay should be carried out within 15 minutes after adding stop solution.

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level E-Cad were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level E-Cad were tested on 3 different plates, 8 replicates in each plate.

CV(%) = SD/meanX100

Intra-Assay: CV<10%

Inter-Assay: CV<12%

Assay range

0.6pg/ml -80pg/ml

Sensitivity:

0.1 pg/ml