Mouse free fatty acid (FFA) ELISA test kit instructions - Database & Sql Blog Articles

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Mouse free fatty acid (FFA) ELISA Test Kit

This kit is for research use only. Not for human or animal diagnostic purposes.

Experimental Principle

The Mouse Free Fatty Acid (FFA) ELISA Kit uses a double-antibody sandwich assay to measure the concentration of FFA in mouse samples. The process involves coating a microtiter plate with a purified mouse FFA antibody, followed by incubation with the sample and HRP-labeled FFA antibody. A complex forms between the antibody, antigen, and enzyme-labeled antibody. After washing, TMB substrate is added, which changes color based on the presence of FFA. The intensity of the color is directly proportional to the FFA concentration in the sample. The absorbance is measured at 450 nm using a microplate reader, and the FFA concentration is calculated from a standard curve.

Kit Composition

  • 1. 30× Washing Solution – 20ml × 1 bottle
  • 2. Enzyme Standard Reagent – 6ml × 1 bottle
  • 3. Enzyme-Labeled Coating Plate – 12 wells × 8 strips
  • 4. Sample Diluent – 6ml × 1 bottle
  • 5. Reagent A – 6ml × 1 bottle
  • 6. Color Developer B – 6ml × 1 bottle
  • 7. Stop Solution – 6ml × 1 bottle
  • 8. Standard (1600μmol/L) – 0.5ml × 1 bottle
  • 9. Standard Dilutions – 1.5ml × 1 bottle
  • 10. Instruction Manual – 1 copy
  • 11. Sealing Film – 2 sheets
  • 12. Sealed Bag – 1

Sample Requirements

1. Samples should be processed as soon as possible after collection. If not used immediately, store at -20°C. Avoid repeated freezing and thawing.

2. Samples containing NaN3 cannot be tested, as it may inhibit HRP activity.

Procedure

  1. Standard Dilution: Prepare a series of standards using the provided diluent. For example, start with 150 μL of the original standard, then dilute stepwise to obtain concentrations ranging from 50 μmol/L to 400 μmol/L.
  2. Loading: Add 50 μL of standard, 40 μL of sample diluent, and 10 μL of sample into each well. Mix gently without touching the well walls.
  3. Incubation: Seal the plate and incubate at 37°C for 30 minutes.
  4. Washing: Wash the plate 5 times with diluted washing solution (1:30), then pat dry.
  5. Add Enzyme: Add 50 μL of enzyme-labeled reagent to each well except the blank control.
  6. Incubation: Incubate again at 37°C for 30 minutes.
  7. Color Development: Add 50 μL of TMB developer to each well and incubate at 37°C for 15 minutes.
  8. Stop Reaction: Add 50 μL of stop solution to terminate the reaction. The color will turn yellow.
  9. Measurement: Read the absorbance at 450 nm within 15 minutes of adding the stop solution.

Calculation

Create a standard curve by plotting the OD values against the known concentrations. Use linear regression to determine the sample concentration. Multiply the result by the dilution factor to obtain the actual concentration.

Precautions

  1. Allow the kit to reach room temperature (15–30 minutes) before use. Store unopened enzyme reagents in a sealed bag.
  2. If the washing solution crystallizes, warm it gently in a water bath before use. It will not affect results.
  3. Use a pipette for accuracy. Limit loading time to 5 minutes per batch. A multi-channel pipette is recommended for large numbers of samples.
  4. Always prepare a standard curve and run duplicates. If the sample OD exceeds the highest standard, dilute the sample before testing and adjust the final calculation accordingly.
  5. Use a new sealing film for each experiment to prevent cross-contamination.
  6. Keep the substrate away from light during storage and handling.
  7. Follow the manual strictly. Results must be confirmed with a microplate reader.
  8. Treat all samples, washes, and waste as biohazardous materials.
  9. Do not mix reagents from different batches.
  10. In case of discrepancies, the English version of the manual takes precedence.

Storage and Shelf Life

  • Store the kit at 2–8°C.
  • Shelf life: 6 months from the date of manufacture.

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