Description
KAPCB Colorimetric Cell-Based ELISA
The KAPCB Colorimetric Cell-Based ELISA Kit offered by Assay Genie is a powerful tool for measuring the activity of kinase-activated protein C (KAPCB) in cell-based assays. This kit provides accurate and reliable results with high sensitivity and specificity, making it ideal for a wide range of research applications.KAPCB is a key regulator of cell signaling pathways and is involved in various cellular processes, including cell growth, proliferation, and differentiation. Dysregulation of KAPCB activity has been implicated in a range of diseases, including cancer, autoimmune disorders, and neurodegenerative conditions, making it an important target for drug development and biomarker discovery.
With the KAPCB Colorimetric Cell-Based ELISA Kit from Assay Genie, researchers can assess KAPCB activity in cell cultures with ease and precision. This versatile kit is suitable for use with a variety of cell types and can provide valuable insights into the mechanisms underlying disease progression and potential therapeutic targets.
| Product Name: | KAPCB Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00988 |
| ELISA Type: | Cell-Based |
| Target: | KAPCB |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The KAPCB Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect KAPCB protein expression profile in cells. The kit can be used for measuring the relative amounts of KAPCB in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on KAPCB.
Qualitative determination of KAPCB concentration is achieved by an indirect ELISA format. In essence, KAPCB is captured by KAPCB-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: 5567, UniProt ID: P22694, OMIM: 176892, Unigene: Hs.487325 |
| Gene Symbol: | PRKACB |
| Sub Type: | None |
| UniProt Protein Function: | PKACB: Mediates cAMP-dependent signaling triggered by receptor binding to GPCRs. PKA activation regulates diverse cellular processes such as cell proliferation, the cell cycle, differentiation and regulation of microtubule dynamics, chromatin condensation and decondensation, nuclear envelope disassembly and reassembly, as well as regulation of intracellular transport mechanisms and ion flux. Regulates the abundance of compartmentalized pools of its regulatory subunits through phosphorylation of PJA2 which binds and ubiquitinates these subunits, leading to their subsequent proteolysis. A number of inactive tetrameric holoenzymes are produced by the combination of homo- or heterodimers of the different regulatory subunits associated with 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. The cAMP-dependent protein kinase catalytic subunit binds PJA2. Isoform 1 is most abundant in the brain, with low level expression in kidney. Isoform 2 is predominantly expressed in thymus, spleen and kidney. Isoform 3 and isoform 4 are only expressed in the brain. Activated by cAMP. Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. cAMP subfamily. 9 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, AGC; EC 2.7.11.11; AGC group; PKA family Chromosomal Location of Human Ortholog: 1p31.1 Cellular Component: nucleoplasm; centrosome; perinuclear region of cytoplasm; plasma membrane; cytosol; cAMP-dependent protein kinase complex Molecular Function:protein binding; cAMP-dependent protein kinase activity; ubiquitin protein ligase binding; magnesium ion binding; ATP 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; glucose metabolic process; pathogenesis; negative regulation of meiotic cell cycle; signal transduction; protein amino acid phosphorylation; gluconeogenesis; G-protein signaling, coupled to cAMP nucleotide second messenger; synaptic transmission; phospholipase C activation; triacylglycerol catabolic process; carbohydrate metabolic process; neural tube closure; energy reserve metabolic process; renal water homeostasis; innate immune response; blood coagulation; transmembrane transport; regulation of insulin secretion |
| NCBI Summary: | The protein encoded by this gene is a member of the serine/threonine protein kinase family. The encoded protein is a catalytic subunit of cAMP (cyclic AMP)-dependent protein kinase, which mediates signalling though cAMP. cAMP signaling is important to a number of processes, including cell proliferaton and differentiation. Multiple alternatively spliced transcript variants encoding distinct isoforms have been observed. [provided by RefSeq, Jul 2014] |
| UniProt Code: | P22694 |
| NCBI GenInfo Identifier: | 125210 |
| NCBI Gene ID: | 5567 |
| NCBI Accession: | P22694.2 |
| UniProt Secondary Accession: | P22694,Q14VH1, Q59GC0, Q5BNE9, Q5BNF0, Q5BNF1, Q5BNF2 Q5BNF3, Q5CZ92, B1APG4, B4DKB0, B4E2Q1, |
| UniProt Related Accession: | P22694 |
| Molecular Weight: | 37,110 Da |
| NCBI Full Name: | cAMP-dependent protein kinase catalytic subunit beta |
| NCBI Synonym Full Names: | protein kinase, cAMP-dependent, catalytic, beta |
| NCBI Official Symbol: | PRKACB |
| NCBI Official Synonym Symbols: | PKACB; PKA C-beta |
| NCBI Protein Information: | cAMP-dependent protein kinase catalytic subunit beta; protein kinase A catalytic subunit beta; cAMP-dependent protein kinase catalytic beta subunit isoform 4ab |
| UniProt Protein Name: | cAMP-dependent protein kinase catalytic subunit beta |
| Protein Family: | cAMP-dependent protein kinase |
| UniProt Gene Name: | PRKACB |
| UniProt Entry Name: | KAPCB_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-KAPCB 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.)
