**Rat Bone Morphogenetic Protein-7 (BMP-7) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed to quantify the levels of bone morphogenetic protein-7 (BMP-7) in rat serum, plasma, urine, cell culture supernatants, and other biological fluids. The method employed is a double-antibody sandwich ELISA, which ensures high specificity and sensitivity.
**Principle of Operation:**
The kit uses a microtiter plate pre-coated with a specific antibody against BMP-7. After adding the sample, BMP-7 binds to the immobilized antibody. A second HRP-conjugated antibody is then added, forming an antibody-antigen-enzyme complex. Following washing steps, TMB substrate is added, which turns blue under HRP catalysis and then changes to yellow upon acid termination. The intensity of the color is directly proportional to the concentration of BMP-7 in the sample. The absorbance is measured at 450 nm using a microplate reader, and the concentration is determined by comparing the sample OD value to a standard curve.
**Kit Components (48-well / 96-well):**
- 1×48 / 1×96 Pre-coated microtiter plate
- 2–8°C storage standards: 0.5 ml × 1 bottle (900 pg/ml)
- Standard diluent: 1.5 ml × 1 bottle
- Enzyme-labeled reagent: 3 ml × 1 bottle
- Sample diluent: 3 ml × 1 bottle
- TMB Developer A & B: 3 ml × 1 bottle each
- Wash buffer (20×): 20 ml × 20 times / 20 ml × 30 times
- Sealing film: 2 pieces
- Storage bag: 1 piece
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use anticoagulants like EDTA or heparin, mix well, and centrifuge as above.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes after collection.
- **Tissue Homogenate:** Weigh the tissue, homogenize in PBS, centrifuge, and collect the supernatant.
**Procedure Summary:**
1. Prepare standard dilutions in duplicate.
2. Add 40 μl sample diluent and 10 μl sample to each well.
3. Seal and incubate at 37°C for 30 minutes.
4. Wash 5 times with wash buffer.
5. Add 50 μl enzyme-labeled reagent.
6. Incubate again for 30 minutes.
7. Wash again.
8. Add 50 μl TMB A and B, incubate for 15 minutes.
9. Stop reaction with 50 μl stop solution.
10. Measure OD at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each run.
- Always prepare a standard curve and consider sample dilution if OD values exceed the standard range.
- Handle all reagents carefully and dispose of waste as biohazardous material.
**Performance Characteristics:**
- Sensitivity: 24 pg/ml
- Range: 24 pg/ml – 800 pg/ml
- Correlation coefficient (R²): ≥ 0.95
- Intra- and inter-assay variability: <9% and <11%, respectively
**Storage & Shelf Life:**
- Store the kit at 2–8°C.
- Valid for 6 months from the date of manufacture.
This kit provides a reliable and accurate method for measuring BMP-7 levels in rat samples, making it ideal for basic research and drug development studies.
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