Description
Amylin Colorimetric Cell-Based ELISA Kit
The Amylin Colorimetric Cell-Based ELISA Kit offered by Assay Genie is specifically designed for the accurate detection of amylin levels in cell culture supernatants. With high sensitivity and specificity, this kit provides reliable and reproducible results, making it ideal for a variety of research applications.Amylin is a peptide hormone that plays a key role in glucose metabolism and appetite regulation. Abnormal amylin levels have been associated with conditions such as diabetes and obesity, making it an important biomarker for studying these diseases and potential therapeutic interventions.
With the Amylin Colorimetric Cell-Based ELISA Kit, researchers can confidently measure amylin levels in various samples, providing valuable insights into the role of this hormone in health and disease. This kit is a valuable tool for advancing research in the fields of endocrinology, metabolic disorders, and drug development.
| Product Name: | Amylin Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00525 |
| ELISA Type: | Cell-Based |
| Target: | Amylin |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The Amylin Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Amylin protein expression profile in cells. The kit can be used for measuring the relative amounts of Amylin in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Amylin.
Qualitative determination of Amylin concentration is achieved by an indirect ELISA format. In essence, Amylin is captured by Amylin-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:
| 1. | A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values. |
| 2. | Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted. |
| Database Information: | Gene ID: 3375, UniProt ID: P10997, OMIM: 147940, Unigene: Hs.46835 |
| Gene Symbol: | IAPP |
| Sub Type: | None |
| UniProt Protein Function: | IAPP: Selectively inhibits insulin-stimulated glucose utilization and glycogen deposition in muscle, while not affecting adipocyte glucose metabolism. Belongs to the calcitonin family. |
| UniProt Protein Details: | Protein type:Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 12p12.1 Cellular Component: extracellular space; cell soma; extracellular region Molecular Function:identical protein binding; hormone activity; receptor binding Biological Process: negative regulation of cell differentiation; cell-cell signaling; eating behavior; apoptosis; sensory perception of pain; negative regulation of bone resorption; signal transduction; endocrine pancreas development |
| NCBI Summary: | The protein encoded by this gene is commonly found in pancreatic islets of patients suffering diabetes mellitus type II, or harboring an insulinoma. Studies suggest that this protein, like the related beta-amyloid (Abeta) associated with Alzheimer's disease, can induce apoptotic cell-death in particular cultured cells, an effect that may be relevant to the development of type II diabetes. This protein also exhibits an bactericidal, antimicrobial activity. [provided by RefSeq, Sep 2014] |
| UniProt Code: | P10997 |
| NCBI GenInfo Identifier: | 124006 |
| NCBI Gene ID: | 3375 |
| NCBI Accession: | P10997.1 |
| UniProt Secondary Accession: | P10997,Q0ZD87, Q14598, |
| UniProt Related Accession: | P10997 |
| Molecular Weight: | 89 |
| NCBI Full Name: | Islet amyloid polypeptide |
| NCBI Synonym Full Names: | islet amyloid polypeptide |
| NCBI Official Symbol: | IAPP |
| NCBI Official Synonym Symbols: | DAP; IAP |
| NCBI Protein Information: | islet amyloid polypeptide; amylin; insulinoma amyloid peptide; diabetes-associated peptide; Islet amyloid polypeptide (diabetes-associated peptide; amylin) |
| UniProt Protein Name: | Islet amyloid polypeptide |
| UniProt Synonym Protein Names: | Amylin; Diabetes-associated peptide; DAP; Insulinoma amyloid peptide |
| Protein Family: | Islet amyloid polypeptide |
| UniProt Gene Name: | IAPP |
| UniProt Entry Name: | IAPP_HUMAN |
| Component | Quantity |
| 96-Well Cell Culture Clear-Bottom Microplate | 2 plates |
| 10X TBS | 24 mL |
| Quenching Buffer | 24 mL |
| Blocking Buffer | 50 mL |
| 15X Wash Buffer | 50 mL |
| Primary Antibody Diluent | 12 mL |
| 100x Anti-Phospho Target Antibody | 60 µL |
| 100x Anti-Target Antibody | 60 µL |
| Anti-GAPDH Antibody | 60 µL |
| HRP-Conjugated Anti-Rabbit IgG Antibody | 12 mL |
| HRP-Conjugated Anti-Mouse IgG Antibody | 12 mL |
| SDS Solution | 12 mL |
| Stop Solution | 24 mL |
| Ready-to-Use Substrate | 12 mL |
| Crystal Violet Solution | 12 mL |
| Adhesive Plate Seals | 2 seals |
The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
- Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
- Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
- 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
- Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
- Graph paper or computer software capable of generating or displaying logarithmic functions
- Absorbent papers or vacuum aspirator
- Test tubes or microfuge tubes capable of storing ≥1 ml
- Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
- Orbital shaker (optional)
- Deionized or sterile water
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1. | Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells. |
| 2. | Incubate the cells for overnight at 37°C, 5% CO2. |
| 3. | Treat the cells as desired. |
| 4. | Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice. |
| 5. | Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells. |
| 6. | Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week. |
| 7. | Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature. |
| 8. | Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time. |
| 9. | Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature. |
| 10. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 11. | Add 50 µL of 1x primary antibodies (Anti-Amylin Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature. |
| 12. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 13. | Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature. |
| 14. | Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time. |
| 15. | Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark. |
| 16. | Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader. |
(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)
