Description
PKA-R2beta (Phospho-Ser113)Colorimetric Cell-Based ELISA Kit
The PKA R2beta Phospho-Ser113 Colorimetric Cell-Based ELISA Kit is a highly sensitive and specific kit designed for the accurate detection of phosphorylated PKA R2beta (Ser113) levels in cell lysates. This kit provides reliable and reproducible results, making it a valuable tool for researchers studying protein kinase A (PKA) signaling pathways.PKA R2beta is a regulatory subunit of PKA, a crucial enzyme involved in various cellular processes, including cell growth, metabolism, and gene expression.
Phosphorylation of PKA R2beta at Ser113 plays a key role in regulating PKA activity and downstream signaling cascades.By accurately measuring phosphorylated PKA R2beta levels, researchers can gain insights into the role of PKA signaling in cellular functions and diseases. This kit is suitable for a wide range of cell-based assays, making it an essential tool for studying PKA signaling pathways and developing potential therapies targeting this pathway.
| Product Name: | PKA-R2beta (Phospho-Ser113)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB01433 |
| ELISA Type: | Cell-Based |
| Target: | PKA-R2beta (Phospho-Ser113) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The PKA-R2beta (Phospho-Ser113) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect PKA-R2beta protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PKA-R2beta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PKA-R2beta phosphorylation.
Qualitative determination of PKA-R2beta (Phospho-Ser113) concentration is achieved by an indirect ELISA format. In essence, PKA-R2beta (Phospho-Ser113) is captured by PKA-R2beta (Phospho-Ser113)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 5577, UniProt ID: P31323, OMIM: 176912, Unigene: Hs.433068 |
| Gene Symbol: | PRKAR2B |
| Sub Type: | Phospho |
| UniProt Protein Function: | PKAR2B: a regulatory subunit of cAMP-regulated protein kinase. The inactive form of the enzyme is composed of two regulatory chains and two catalytic chains. Activation by cAMP produces two active catalytic monomers and a regulatory dimer that binds four cAMP molecules. Four types of regulatory chains are found: I-alpha, I-beta, II-alpha, and II-beta. Their expression varies among tissues and is in some cases constitutive and in others inducible. Type II regulatory chains mediate membrane association by binding to anchoring proteins, including the MAP2 kinase. |
| UniProt Protein Details: | Protein type:Mitochondrial; Protein kinase, regulatory subunit Chromosomal Location of Human Ortholog: 7q22.3 Cellular Component: centrosome; cytoplasm; cytosol; dendritic shaft; dendritic spine; plasma membrane Molecular Function:cAMP-dependent protein kinase inhibitor activity; cAMP-dependent protein kinase regulator activity; protein binding; ubiquitin protein ligase binding Biological Process: activation of protein kinase A; blood coagulation; G2/M transition of mitotic cell cycle; renal water homeostasis |
| NCBI Summary: | cAMP is a signaling molecule important for a variety of cellular functions. cAMP exerts its effects by activating the cAMP-dependent protein kinase, which transduces the signal through phosphorylation of different target proteins. The inactive kinase holoenzyme is a tetramer composed of two regulatory and two catalytic subunits. cAMP causes the dissociation of the inactive holoenzyme into a dimer of regulatory subunits bound to four cAMP and two free monomeric catalytic subunits. Four different regulatory subunits and three catalytic subunits have been identified in humans. The protein encoded by this gene is one of the regulatory subunits. This subunit can be phosphorylated by the activated catalytic subunit. This subunit has been shown to interact with and suppress the transcriptional activity of the cAMP responsive element binding protein 1 (CREB1) in activated T cells. Knockout studies in mice suggest that this subunit may play an important role in regulating energy balance and adiposity. The studies also suggest that this subunit may mediate the gene induction and cataleptic behavior induced by haloperidol. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P31323 |
| NCBI GenInfo Identifier: | 206729918 |
| NCBI Gene ID: | 5577 |
| NCBI Accession: | P31323.3 |
| UniProt Secondary Accession: | P31323,A4D0R9, |
| UniProt Related Accession: | P31323 |
| Molecular Weight: | 46kDa |
| NCBI Full Name: | cAMP-dependent protein kinase type II-beta regulatory subunit |
| NCBI Synonym Full Names: | protein kinase cAMP-dependent type II regulatory subunit beta |
| NCBI Official Symbol: | PRKAR2B |
| NCBI Official Synonym Symbols: | PRKAR2; RII-BETA |
| NCBI Protein Information: | cAMP-dependent protein kinase type II-beta regulatory subunit |
| UniProt Protein Name: | cAMP-dependent protein kinase type II-beta regulatory subunit |
| Protein Family: | cAMP-dependent protein kinase |
| UniProt Gene Name: | PRKAR2B |
| 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-PKA-R2beta (Phospho-Ser113) Antibody, Anti-PKA-R2beta 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.)
