Description
AKAP5 Colorimetric Cell-Based ELISA
The AKAP5 Colorimetric Cell-Based ELISA Kit is a cutting-edge assay designed for the accurate and reliable detection of AKAP5 levels in cell lysates and tissue samples. This kit offers high sensitivity and specificity, allowing for precise quantification of AKAP5 protein levels in various research applications.AKAP5, also known as A-kinase anchor protein 5, plays a crucial role in regulating intracellular signaling pathways and protein kinase A (PKA) activity. Dysregulation of AKAP5 has been implicated in various diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions.
Therefore, studying AKAP5 levels can provide valuable insights into disease mechanisms and potential therapeutic targets.The AKAP5 Colorimetric Cell-Based ELISA Kit is easy to use, providing researchers with a simple yet powerful tool for studying AKAP5 expression and function in cellular processes. With this kit, researchers can accelerate their research and advance our understanding of AKAP5 biology.
| Product Name: | AKAP5 Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB00992 |
| ELISA Type: | Cell-Based |
| Target: | AKAP5 |
| Reactivity: | Human |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nmStorage/Stability:4°C/6 Months |
| Format: | 96-Well Microplate |
The AKAP5 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect AKAP5 protein expression profile in cells. The kit can be used for measuring the relative amounts of AKAP5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on AKAP5.
Qualitative determination of AKAP5 concentration is achieved by an indirect ELISA format. In essence, AKAP5 is captured by AKAP5-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: 9495, UniProt ID: P24588, OMIM: 604688, Unigene: Hs.656683 |
| Gene Symbol: | AKAP5 |
| Sub Type: | None |
| UniProt Protein Function: | AKAP5: May anchor the PKA protein to cytoskeletal and/or organelle-associated proteins, targeting the signal carried by cAMP to specific intracellular effectors. Association with to the beta2-adrenergic receptor (beta2-AR) not only regulates beta2-AR signaling pathway, but also the activation by PKA by switching off the beta2-AR signaling cascade. Binding protein for dimer of the RII-beta regulatory subunit of cAMP-dependent protein kinase (PKA) and also for the protein kinase C (PKC) and the phosphatase calcineurin (PP2B). Each enzyme is inhibited when bound to the anchoring protein. Also binds the beta2-adrenergic receptor. Part of a complex containing AKAP5, ADCY5, ADCY6 AND PDE4C. Interacts with ADCY8, and enhances its phosphorylation at lipid rafts. Predominantly in the cerebral cortex and the postsynaptic densities of the forebrain, and to a lesser extent in adrenal medulla, lung and anterior pituitary. |
| UniProt Protein Details: | Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 14q23.3 Cellular Component: cytosol; plasma membrane Molecular Function:adenylate cyclase binding; protein binding; protein kinase A binding Biological Process: positive regulation of cAMP biosynthetic process; synaptic transmission |
| NCBI Summary: | The A-kinase anchor proteins (AKAPs) are a group of structurally diverse proteins, which have the common function of binding to the regulatory subunit of protein kinase A (PKA) and confining the holoenzyme to discrete locations within the cell. This gene encodes a member of the AKAP family. The encoded protein binds to the RII-beta regulatory subunit of PKA, and also to protein kinase C and the phosphatase calcineurin. It is predominantly expressed in cerebral cortex and may anchor the PKA protein at postsynaptic densities (PSD) and be involved in the regulation of postsynaptic events. It is also expressed in T lymphocytes and may function to inhibit interleukin-2 transcription by disrupting calcineurin-dependent dephosphorylation of NFAT. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P24588 |
| NCBI GenInfo Identifier: | 281185503 |
| NCBI Gene ID: | 9495 |
| NCBI Accession: | P24588.3 |
| UniProt Secondary Accession: | P24588,A2RRB8, |
| UniProt Related Accession: | P24588 |
| Molecular Weight: | 47,088 Da |
| NCBI Full Name: | A-kinase anchor protein 5 |
| NCBI Synonym Full Names: | A-kinase anchoring protein 5 |
| NCBI Official Symbol: | AKAP5 |
| NCBI Official Synonym Symbols: | H21; AKAP75; AKAP79 |
| NCBI Protein Information: | A-kinase anchor protein 5 |
| UniProt Protein Name: | A-kinase anchor protein 5 |
| UniProt Synonym Protein Names: | A-kinase anchor protein 79 kDa; AKAP 79; H21; cAMP-dependent protein kinase regulatory subunit II high affinity-binding protein |
| Protein Family: | A-kinase anchor protein |
| UniProt Gene Name: | AKAP5 |
| UniProt Entry Name: | AKAP5_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-AKAP5 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.)
