Description
ADD2 Colorimetric Cell-Based ELISA Kit
The Add2 Colorimetric Cell-Based ELISA Kit is a versatile and reliable tool for detecting and quantifying human angiogenin levels in various sample types, including serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for a wide range of research applications.Angiogenin is a key protein involved in angiogenesis, the process of new blood vessel formation, as well as cell proliferation. This protein plays a crucial role in various health conditions such as cancer, cardiovascular diseases, and neurodegenerative disorders, making it an important biomarker for studying and potentially treating these conditions.
With the Add2 Colorimetric Cell-Based ELISA Kit, researchers can confidently measure angiogenin levels in their samples, leading to valuable insights and advancements in the field of angiogenesis research and related diseases. This kit is a valuable addition to any laboratory conducting studies on angiogenin and its roles in health and disease.
| Product Name: | ADD2 Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00515 |
| ELISA Type: | Cell-Based |
| Target: | ADD2 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The ADD2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ADD2 protein expression profile in cells. The kit can be used for measuring the relative amounts of ADD2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ADD2.
Qualitative determination of ADD2 concentration is achieved by an indirect ELISA format. In essence, ADD2 is captured by ADD2-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: 119, UniProt ID: P35612, OMIM: 102681, Unigene: Hs.188528 |
| Gene Symbol: | ADD2 |
| Sub Type: | None |
| UniProt Protein Function: | ADD2: a cytoskeletal protein that promotes the assembly of the spectrin-actin network. Adducin is a heterodimeric protein that consists of related subunits. Alpha- and beta-adducin include a protease-resistant N-terminal region and a protease-sensitive, hydrophilic C-terminal region. Adducin binds with high affinity to Ca(2+)/calmodulin and is a substrate for protein kinases A and C. Beta-adducin is restricted to brain and hematopoietic tissues. Three alternatively-spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:Cytoskeletal; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 2p13.3 Cellular Component: cytoplasmic membrane-bound vesicle; cytosol; F-actin capping protein complex; plasma membrane Molecular Function:actin binding; actin filament binding; calmodulin binding; protein heterodimerization activity; protein homodimerization activity; spectrin binding Biological Process: actin cytoskeleton organization and biogenesis; actin filament bundle formation; barbed-end actin filament capping; ion transport; positive regulation of protein binding; protein complex assembly; transmembrane transport |
| NCBI Summary: | Adducins are heteromeric proteins composed of different subunits referred to as adducin alpha, beta and gamma. The three subunits are encoded by distinct genes and belong to a family of membrane skeletal proteins involved in the assembly of spectrin-actin network in erythrocytes and at sites of cell-cell contact in epithelial tissues. While adducins alpha and gamma are ubiquitously expressed, the expression of adducin beta is restricted to brain and hematopoietic tissues. Adducin, originally purified from human erythrocytes, was found to be a heterodimer of adducins alpha and beta. Polymorphisms resulting in amino acid substitutions in these two subunits have been associated with the regulation of blood pressure in an animal model of hypertension. Heterodimers consisting of alpha and gamma subunits have also been described. Structurally, each subunit is comprised of two distinct domains. The amino-terminal region is protease resistant and globular in shape, while the carboxy-terminal region is protease sensitive. The latter contains multiple phosphorylation sites for protein kinase C, the binding site for calmodulin, and is required for association with spectrin and actin. Alternatively spliced transcript variants have been described. [provided by RefSeq, Jun 2010] |
| UniProt Code: | P35612 |
| NCBI GenInfo Identifier: | 543774 |
| NCBI Gene ID: | 119 |
| NCBI Accession: | P35612.3 |
| UniProt Secondary Accession: | P35612,Q13482, Q16412, Q59G82, Q5U5P4, Q6P0P2, Q6PGQ4 Q7Z688, A8K4P2, B4DM17, D6W5G7, D6W5G8, |
| UniProt Related Accession: | P35612 |
| Molecular Weight: | 64,217 Da |
| NCBI Full Name: | Beta-adducin |
| NCBI Synonym Full Names: | adducin 2 |
| NCBI Official Symbol: | ADD2 |
| NCBI Official Synonym Symbols: | ADDB |
| NCBI Protein Information: | beta-adducin |
| UniProt Protein Name: | Beta-adducin |
| UniProt Synonym Protein Names: | Erythrocyte adducin subunit beta |
| Protein Family: | Beta-adducin |
| UniProt Gene Name: | ADD2 |
| UniProt Entry Name: | ADDB_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-ADD2 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.)
