Description
CD88/C5aR Colorimetric Cell-Based ELISA Kit
The CD88/C5aR Colorimetric Cell-Based ELISA Kit from Assay Genie is a powerful tool for researchers interested in studying the CD88/C5aR pathway. This kit allows for the accurate detection of CD88/C5aR levels in cell lysates and samples, with high sensitivity and specificity, ensuring reliable and reproducible results.The CD88/C5aR pathway is involved in inflammatory responses, immune regulation, and cell migration, making it a key target for research in a variety of disease conditions such as autoimmune disorders, inflammatory diseases, and cancer.
By accurately measuring CD88/C5aR levels, researchers can gain valuable insights into the mechanisms underlying these diseases and potentially identify new therapeutic targets.With its user-friendly protocol and high-quality reagents, the CD88/C5aR Colorimetric Cell-Based ELISA Kit is a valuable addition to any research laboratory looking to explore the role of the CD88/C5aR pathway in health and disease.
| Product Name: | CD88/C5aR Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00572 |
| ELISA Type: | Cell-Based |
| Target: | CD88/C5aR |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The CD88/C5aR Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CD88/C5aR protein expression profile in cells. The kit can be used for measuring the relative amounts of CD88/C5aR in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CD88/C5aR.
Qualitative determination of CD88/C5aR concentration is achieved by an indirect ELISA format. In essence, CD88/C5aR is captured by CD88/C5aR-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: 728, UniProt ID: P21730, OMIM: 113995, Unigene: Hs.2161 |
| Gene Symbol: | C5AR1 |
| Sub Type: | None |
| UniProt Protein Function: | C5aR: a family 1 G-protein coupled receptor that stimulates the GTPase activity of Gi2. Receptor for the chemotactic and inflammatory complement factor C5a. Stimulates chemotaxis, granule enzyme release and superoxide anion production. Phosphorylated in response to C5a or after stimulation of cells with phorbol esters. Expressed on monocytes, granulocytes, dendritic cells, astrocytes and microglia. |
| UniProt Protein Details: | Protein type:Membrane protein, integral; Receptor, GPCR; GPCR, family 1; Membrane protein, multi-pass Chromosomal Location of Human Ortholog: 19q13.3-q13.4 Cellular Component: cell surface; basolateral plasma membrane; integral to plasma membrane; apical part of cell; cytoplasmic membrane-bound vesicle; plasma membrane Molecular Function:complement component C5a binding; complement component C5a receptor activity; C5a anaphylatoxin receptor activity Biological Process: neutrophil chemotaxis; sensory perception of chemical stimulus; organ regeneration; apoptosis; activation of MAPK activity; response to lipopolysaccharide; cell proliferation in hindbrain; signal transduction; chemotaxis; mRNA transcription from RNA polymerase II promoter; defense response to Gram-positive bacterium; elevation of cytosolic calcium ion concentration; phospholipase C activation; response to peptidoglycan; cellular defense response; immune response; negative regulation of neuron apoptosis; inflammatory response; positive regulation of epithelial cell proliferation |
| UniProt Code: | P21730 |
| NCBI GenInfo Identifier: | 311033355 |
| NCBI Gene ID: | 728 |
| NCBI Accession: | P21730.2 |
| UniProt Related Accession: | P21730 |
| Molecular Weight: | 39,336 Da |
| NCBI Full Name: | C5a anaphylatoxin chemotactic receptor 1 |
| NCBI Synonym Full Names: | complement component 5a receptor 1 |
| NCBI Official Symbol: | C5AR1 |
| NCBI Official Synonym Symbols: | C5A; C5AR; C5R1; CD88 |
| NCBI Protein Information: | C5a anaphylatoxin chemotactic receptor 1; C5a-R; C5a ligand; C5a anaphylatoxin receptor; complement component 5 receptor 1 |
| UniProt Protein Name: | C5a anaphylatoxin chemotactic receptor 1 |
| UniProt Synonym Protein Names: | C5a anaphylatoxin chemotactic receptor; C5a-R; C5aR; CD_antigen: CD88 |
| Protein Family: | C5a anaphylatoxin chemotactic receptor |
| UniProt Gene Name: | C5AR1 |
| UniProt Entry Name: | C5AR1_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-CD88/C5aR 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.)
