KAP0 Colorimetric Cell-Based ELISA
- SKU:
- CBCAB00974
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | KAP0 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00974 |
| ELISA Type: | Cell-Based |
| Target: | KAP0 |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The KAP0 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect KAP0 protein expression profile in cells. The kit can be used for measuring the relative amounts of KAP0 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on KAP0.
Qualitative determination of KAP0 concentration is achieved by an indirect ELISA format. In essence, KAP0 is captured by KAP0-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: 5573, UniProt ID: P10644, OMIM: 188830, Unigene: Hs.280342 |
| Gene Symbol: | PRKAR1A |
| Sub Type: | None |
| UniProt Protein Function: | PKAR1A: 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. Interacts with RFC2: the complex may be involved in cell survival. Defects in PRKAR1A are the cause of Carney complex type 1 (CNC1). CNC is a multiple neoplasia syndrome characterized by spotty skin pigmentation, cardiac and other myxomas, endocrine tumors, and psammomatous melanotic schwannomas. |
| UniProt Protein Details: | Protein type:Protein kinase, regulatory subunit Chromosomal Location of Human Ortholog: 17q24.2 Cellular Component: protein complex; membrane; plasma membrane; neuromuscular junction; cytosol; AMP-activated protein kinase complex; cAMP-dependent protein kinase complex Molecular Function:protein binding; ubiquitin protein ligase binding; cAMP-dependent protein kinase inhibitor activity; cAMP-dependent protein kinase regulator activity; cAMP binding Biological Process: epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; nerve growth factor receptor signaling pathway; water transport; activation of protein kinase A; female meiosis; cardiac muscle cell proliferation; sarcomere organization; signal transduction; regulation of transcription from RNA polymerase II promoter; mesoderm formation; phospholipase C activation; negative regulation of meiosis; energy reserve metabolic process; innate immune response; renal water homeostasis; blood coagulation; regulation of insulin secretion; transmembrane transport Disease: Myxoma, Intracardiac; Carney Complex, Type 1; Thyroid Carcinoma, Papillary; Acrodysostosis 1 With Or Without Hormone Resistance; Pigmented Nodular Adrenocortical Disease, Primary, 1 |
| 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. This gene encodes one of the regulatory subunits. This protein was found to be a tissue-specific extinguisher that down-regulates the expression of seven liver genes in hepatoma x fibroblast hybrids. Mutations in this gene cause Carney complex (CNC). This gene can fuse to the RET protooncogene by gene rearrangement and form the thyroid tumor-specific chimeric oncogene known as PTC2. A nonconventional nuclear localization sequence (NLS) has been found for this protein which suggests a role in DNA replication via the protein serving as a nuclear transport protein for the second subunit of the Replication Factor C (RFC40). Several alternatively spliced transcript variants encoding two different isoforms have been observed. [provided by RefSeq, Jan 2013] |
| UniProt Code: | P10644 |
| NCBI GenInfo Identifier: | 125193 |
| NCBI Gene ID: | 5573 |
| NCBI Accession: | P10644.1 |
| UniProt Secondary Accession: | P10644,Q567S7, K7ER48, |
| UniProt Related Accession: | P10644 |
| Molecular Weight: | 42,982 Da |
| NCBI Full Name: | cAMP-dependent protein kinase type I-alpha regulatory subunit |
| NCBI Synonym Full Names: | protein kinase, cAMP-dependent, regulatory, type I, alpha |
| NCBI Official Symbol: | PRKAR1A |
| NCBI Official Synonym Symbols: | CAR; CNC; CNC1; PKR1; TSE1; ADOHR; PPNAD1; PRKAR1; ACRDYS1 |
| NCBI Protein Information: | cAMP-dependent protein kinase type I-alpha regulatory subunit; Carney complex type 1; tissue-specific extinguisher 1; protein kinase A type 1a regulatory subunit; cAMP-dependent protein kinase regulatory subunit RIalpha; cAMP-dependent protein kinase type I-alpha regulatory chain |
| UniProt Protein Name: | cAMP-dependent protein kinase type I-alpha regulatory subunit |
| UniProt Synonym Protein Names: | Tissue-specific extinguisher 1 |
| Protein Family: | CD83 antigen |
| UniProt Gene Name: | PRKAR1A |
| UniProt Entry Name: | KAP0_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-KAP0 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.)
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