Description
GRK5 Colorimetric Cell-Based ELISA
The GRK5 Colorimetric Cell-Based ELISA Kit is a powerful tool for studying G protein-coupled receptor kinase 5 (GRK5) activity in cells. This kit allows for the accurate measurement of GRK5 levels in cell lysates, providing researchers with valuable insights into the role of this kinase in cell signaling and regulation.GRK5 is a key regulator of G protein-coupled receptor signaling, playing a critical role in numerous physiological processes. Dysregulation of GRK5 activity has been linked to various diseases, including heart failure, diabetes, and neurodegenerative disorders. The GRK5 Colorimetric Cell-Based ELISA Kit offers high sensitivity and specificity, enabling researchers to accurately quantify GRK5 levels and investigate its impact on cellular function.
Whether studying the molecular mechanisms of disease or exploring potential therapeutic targets, the GRK5 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers seeking to advance their understanding of GRK5 biology. With its reliable and reproducible results, this kit is ideal for a wide range of research applications and holds great promise for uncovering new insights into the role of GRK5 in health and disease.
| Product Name: | GRK5 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01002 |
| ELISA Type: | Cell-Based |
| Target: | GRK5 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The GRK5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GRK5 protein expression profile in cells. The kit can be used for measuring the relative amounts of GRK5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GRK5.
Qualitative determination of GRK5 concentration is achieved by an indirect ELISA format. In essence, GRK5 is captured by GRK5-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: 2869, UniProt ID: P34947, OMIM: 600870, Unigene: Hs.524625 |
| Gene Symbol: | GRK5 |
| Sub Type: | None |
| UniProt Protein Function: | GRK5: Serine/threonine kinase that phosphorylates preferentially the activated forms of a variety of G-protein- coupled receptors (GPCRs). Such receptor phosphorylation initiates beta-arrestin-mediated receptor desensitization, internalization, and signaling events leading to their down-regulation. Phosphorylates a variety of GPCRs, including adrenergic receptors, muscarinic acetylcholine receptors (more specifically Gi-coupled M2/M4 subtypes), dopamine receptors and opioid receptors. In addition to GPCRs, also phosphorylates various substrates: Hsc70- interacting protein/ST13, TP53/p53, HDAC5, and arrestin-1/ARRB1. Phosphorylation of ARRB1 by GRK5 inhibits G-protein independent MAPK1/MAPK3 signaling downstream of 5HT4-receptors. Phosphorylation of HDAC5, a repressor of myocyte enhancer factor 2 (MEF2) leading to nuclear export of HDAC5 and allowing MEF2- mediated transcription. Phosphorylation of TP53/p53, a crucial tumor suppressor, inhibits TP53/p53-mediated apoptosis. Phosphorylation of ST13 regulates internalization of the chemokine receptor. Phosphorylates rhodopsin (RHO) (in vitro) and a non G- protein-coupled receptor, LRP6 during Wnt signaling (in vitro). Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. GPRK subfamily. |
| UniProt Protein Details: | Protein type:Kinase, protein; Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.16; AGC group; GRK family; GRK subfamily Chromosomal Location of Human Ortholog: 10q26.11 Cellular Component: cytoplasm; focal adhesion; nuclear membrane; nucleus; plasma membrane Molecular Function:beta-adrenergic receptor kinase activity; phospholipid binding; protein binding; protein kinase C binding; protein serine/threonine kinase activity Biological Process: G-protein signaling, coupled to cAMP nucleotide second messenger; negative regulation of apoptosis; positive regulation of cell proliferation; protein amino acid autophosphorylation; regulation of cell cycle; regulation of G-protein coupled receptor protein signaling pathway; tachykinin signaling pathway |
| NCBI Summary: | This gene encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase subfamily of the Ser/Thr protein kinase family. The protein phosphorylates the activated forms of G protein-coupled receptors thus initiating their deactivation. It has also been shown to play a role in regulating the motility of polymorphonuclear leukocytes (PMNs). [provided by RefSeq, Jul 2008] |
| UniProt Code: | P34947 |
| NCBI GenInfo Identifier: | 462203 |
| NCBI Gene ID: | 2869 |
| NCBI Accession: | P34947.1 |
| UniProt Secondary Accession: | P34947,Q5T059, D3DRD0, |
| UniProt Related Accession: | P34947 |
| Molecular Weight: | 67,787 Da |
| NCBI Full Name: | G protein-coupled receptor kinase 5 |
| NCBI Synonym Full Names: | G protein-coupled receptor kinase 5 |
| NCBI Official Symbol: | GRK5 |
| NCBI Official Synonym Symbols: | GPRK5 |
| NCBI Protein Information: | G protein-coupled receptor kinase 5 |
| UniProt Protein Name: | G protein-coupled receptor kinase 5 |
| UniProt Synonym Protein Names: | G protein-coupled receptor kinase GRK5 |
| Protein Family: | G protein-coupled receptor kinase |
| UniProt Gene Name: | GRK5 |
| UniProt Entry Name: | GRK5_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-GRK5 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.)
