Description
ACVL1 Colorimetric Cell-Based ELISA
The Human ACVL1 Colorimetric Cell-Based ELISA Kit is a comprehensive solution for the precise measurement of ACVL1 levels in various biological samples, including serum, plasma, and cell culture supernatants. With its exceptional sensitivity and specificity, this kit ensures accurate and consistent results, meeting the demands of diverse research applications.ACVL1, also known as acyl-CoA thioesterase 13, plays a crucial role in lipid metabolism and energy homeostasis.
Dysregulation of ACVL1 has been linked to metabolic disorders, obesity, and cardiovascular diseases, highlighting its significance as a potential therapeutic target and biomarker. The ACVL1 Colorimetric Cell-Based ELISA Kit provides researchers with a valuable tool for investigating the role of ACVL1 in health and disease, ultimately advancing our understanding of metabolic disorders and paving the way for novel treatment strategies.
| Product Name: | ACVL1 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00380 |
| ELISA Type: | Cell-Based |
| Target: | ACVL1 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The ACVL1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ACVL1 protein expression profile in cells. The kit can be used for measuring the relative amounts of ACVL1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ACVL1.
Qualitative determination of ACVL1 concentration is achieved by an indirect ELISA format. In essence, ACVL1 is captured by ACVL1-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: 94, UniProt ID: P37023, OMIM: 600376/601284, Unigene: Hs.591026 |
| Gene Symbol: | ACVRL1 |
| Sub Type: | None |
| UniProt Protein Function: | ALK1: a kinase of the STKR family. A type I cell-surface receptor for the TGF-beta superfamily of ligands including Activin A. Highly expressed in human placenta and lung. Deficiency causes hemorrhagic telangiectasia type 2 also known as Rendu-Osler-Weber syndrome 2. |
| UniProt Protein Details: | Protein type:EC 2.7.11.30; Kinase, protein; Membrane protein, integral; Protein kinase, Ser/Thr (receptor); Protein kinase, TKL; STKR family; TKL group; Type1 subfamily Chromosomal Location of Human Ortholog: 12q13.13 Cellular Component: cell surface; integral to plasma membrane; plasma membrane Molecular Function:activin binding; activin receptor activity, type I; ATP binding; protein binding; protein kinase binding; protein serine/threonine kinase activity; SMAD binding; transforming growth factor beta binding; transforming growth factor beta receptor activity; transforming growth factor beta receptor activity, type I Biological Process: angiogenesis; blood circulation; blood vessel endothelial cell proliferation during sprouting angiogenesis; blood vessel maturation; blood vessel remodeling; BMP signaling pathway; lymphangiogenesis; negative regulation of blood vessel endothelial cell migration; negative regulation of cell adhesion; negative regulation of cell growth; negative regulation of cell migration; negative regulation of cell proliferation; negative regulation of focal adhesion formation; positive regulation of BMP signaling pathway; positive regulation of chondrocyte differentiation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein amino acid phosphorylation; regulation of blood pressure; regulation of blood vessel endothelial cell migration; regulation of DNA replication; regulation of endothelial cell proliferation; regulation of transcription, DNA-dependent; signal transduction; transforming growth factor beta receptor signaling pathway; wound healing, spreading of epidermal cells Disease: Telangiectasia, Hereditary Hemorrhagic, Type 2 |
| NCBI Summary: | This gene encodes a type I cell-surface receptor for the TGF-beta superfamily of ligands. It shares with other type I receptors a high degree of similarity in serine-threonine kinase subdomains, a glycine- and serine-rich region (called the GS domain) preceding the kinase domain, and a short C-terminal tail. The encoded protein, sometimes termed ALK1, shares similar domain structures with other closely related ALK or activin receptor-like kinase proteins that form a subfamily of receptor serine/threonine kinases. Mutations in this gene are associated with hemorrhagic telangiectasia type 2, also known as Rendu-Osler-Weber syndrome 2. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P37023 |
| NCBI GenInfo Identifier: | 3915750 |
| NCBI Gene ID: | 94 |
| NCBI Accession: | P37023.2 |
| UniProt Secondary Accession: | P37023,A6NGA8, |
| UniProt Related Accession: | P37023 |
| Molecular Weight: | 56,124 Da |
| NCBI Full Name: | Serine/threonine-protein kinase receptor R3 |
| NCBI Synonym Full Names: | activin A receptor like type 1 |
| NCBI Official Symbol: | ACVRL1 |
| NCBI Official Synonym Symbols: | HHT; ALK1; HHT2; ORW2; SKR3; ALK-1; TSR-I; ACVRLK1 |
| NCBI Protein Information: | serine/threonine-protein kinase receptor R3 |
| UniProt Protein Name: | Serine/threonine-protein kinase receptor R3 |
| UniProt Synonym Protein Names: | Activin receptor-like kinase 1; ALK-1; TGF-B superfamily receptor type I; TSR-I |
| Protein Family: | Serine/threonine-protein kinase haspin |
| UniProt Gene Name: | ACVRL1 |
| 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-ACVL1 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.)
