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User Manual

ELK02J0986, Rat Protein Kinase B (PKB)

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1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.
   Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C).
   Materials supplied
   Name 96 determinations 48 determinations
   Microelisa stripplate 8*12strips 8*6strips
   Standard 0.3ml*6tubes 0.3ml*6tubes
   Sample Diluent 6.0ml 3.0ml
   HRP-Conjugate reagent 10.0ml 5.0ml
   20X Wash solution 25ml 15ml
   Chromogen Solution A 6.0ml 3.0ml
   Chromogen Solution B 6.0ml 3.0ml
   Stop Solution 6.0ml 3.0ml
   Closure plate
   membrane 2 2
   User manual 1 1
   Note: Standard (S0 ￫S5) concentration was followed by:0,2,4,8,16,32 ng/mL.
   Reagent preparation
   20×wash solution:Dilute with Distilled or deionized water 1:20.
   Assay procedure
4. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
5. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
6. Add Sample: Add testing sample 10μl then add 40μl of Sample Diluent to testing sample wel.
7. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
8. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove
   any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
9. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
10. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
    8 Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
    Calculation of results
11. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.
12. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.
13. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.
14. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.
15. The sensitivity by this assay is 0.1 ng/mL.
16. Standard curve.
    Storage: 2-8℃. Validity:six months.
    FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!