Human phosphorylated Akt protein (P-Akt) ELISA kit professional technology test - Database & Sql Blog Articles

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Kaixin micro test

Test probe P100-M3

Specializing in the production of LED chip LED lamp beads

Human phosphorylated Akt protein (P-Akt)

ELISA

Kit

Professional technical survey

This kit is for research use only.

Experimental Principle

The Human Phosphorylated Akt (P-Akt) ELISA Kit is designed to quantitatively measure the levels of phosphorylated Akt protein in biological samples. The method used is a double-antibody sandwich ELISA, where a solid-phase antibody specific to P-Akt is coated onto a microplate. After incubation with the sample, the target antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following washing steps, TMB substrate is introduced, which changes color in the presence of HRP. The intensity of the color is directly proportional to the concentration of P-Akt in the sample. The optical density (OD) is measured at 450 nm, and the concentration is determined using a standard curve.

Composition

- 130x Washing Solution: 20ml × 1 bottle

- Stop Solution: 6ml × 1 bottle

- Enzyme Standard Reagent: 6ml × 1 bottle

- Standard (180ng/L): 0.5ml × 1 bottle

- Enzyme-Labeled Coating Plate: 12 wells × 8

- Sample Diluent: 6ml × 1 bottle

- TMB Substrate A: 6ml × 1 bottle

- TMB Substrate B: 6ml × 1 bottle

- Standard Dilutions: 1.5ml × 1 bottle

- Instruction Manual: 1 copy

- Sealing Film: 2 sheets

- Seal Bag: 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freeze-thaw cycles.

2. Samples containing NaN3 are not suitable, as it may inhibit HRP activity.

Testing Procedure

1. Standard Dilution: Prepare serial dilutions from the original standard according to the provided table.

2. Sample Preparation: Load 50μl of standard, 40μl of sample diluent, and 10μl of sample into each well (final dilution factor 5).

3. Incubate the plate at 37°C for 30 minutes.

4. Wash the plate 5 times with diluted washing solution (1:30).

5. Add 50μl of enzyme-labeled reagent to each well except blank wells.

6. Incubate again for 30 minutes at 37°C.

7. Wash the plate again 5 times.

8. Add 50μl of TMB A and 50μl of TMB B, incubate at 37°C for 15 minutes.

9. Add 50μl of stop solution to terminate the reaction.

10. Measure OD at 450nm within 15 minutes of stopping the reaction.

Calculation

Plot a standard curve using the OD values of standards. Determine the sample concentration based on the curve and multiply by the dilution factor. Alternatively, calculate the linear regression equation and substitute the sample OD value for accurate results.

Precautions

1. Allow the kit to reach room temperature before use. Store unopened enzyme labels in a sealed bag.

2. Wash solution may crystallize; warm it gently if needed.

3. Use a pipette for accuracy. Limit loading time to 5 minutes.

4. Always make a standard curve and consider diluting high-concentration samples if necessary.

5. Single-use sealing film to prevent cross-contamination.

6. Keep substrates away from light.

7. Follow the manual strictly. Results must be confirmed by a microplate reader.

8. Treat all waste as biohazardous material.

9. Do not mix components from different batches.

10. In case of discrepancies, the English manual takes precedence.

Storage Conditions & Expiration

1. Store at 2–8°C.

2. Shelf life: 6 months.