Description
HDAC5 (Phospho-Ser259)Colorimetric Cell-Based ELISA Kit
The HDAC5 Phospho-Ser259 Colorimetric Cell-Based ELISA Kit is a powerful tool for measuring levels of HDAC5 phosphorylated at serine 259 in cell lysates. This kit offers high sensitivity and specificity, providing accurate and reproducible results for a variety of research applications.HDAC5, when phosphorylated at serine 259, plays a crucial role in epigenetic regulation, gene expression, and cellular processes such as apoptosis and cell cycle progression. Dysregulation of HDAC5 phosphorylation has been implicated in diseases such as cancer, neurodegenerative disorders, and cardiovascular diseases, highlighting the importance of studying this biomarker for potential therapeutic interventions.
With easy-to-follow protocols and reliable performance, the HDAC5 Phospho-Ser259 Colorimetric Cell-Based ELISA Kit is an essential tool for researchers looking to understand the role of HDAC5 phosphorylation in disease pathogenesis and identify potential targets for drug development.
| Product Name: | HDAC5 (Phospho-Ser259)Colorimetric Cell-Based ELISA Kit |
| Product Code: | CBCAB00230 |
| ELISA Type: | Cell-Based |
| Target: | HDAC5 (Phospho-Ser259) |
| Reactivity: | Human, Mouse |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The HDAC5 (Phospho-Ser259) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HDAC5 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HDAC5 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on HDAC5 phosphorylation.
Qualitative determination of HDAC5 (Phospho-Ser259) concentration is achieved by an indirect ELISA format. In essence, HDAC5 (Phospho-Ser259) is captured by HDAC5 (Phospho-Ser259)-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: 10014/9734, UniProt ID: Q9UQL6, OMIM: 605315, Unigene: Hs.438782 |
| Gene Symbol: | HDAC5/HDAC9 |
| Sub Type: | Phospho |
| UniProt Protein Function: | HDAC5: a transcriptional regulator of the histone deacetylase family, subfamily 2. Deacetylates lysine residues on the N-terminal part of the core histones H2A, H2B, H3 AND H4. Plays an important role in transcriptional regulation, cell cycle progression and developmental events. Coimmunoprecipitates only with HDAC3 family members. Interacts with myocyte enhancer factor-2 (MEF2) proteins, resulting in repression of MEF2-dependent genes. Two alternatively spliced isoforms have been described. |
| UniProt Protein Details: | Protein type:EC 3.5.1.98; Hydrolase Chromosomal Location of Human Ortholog: 17q21 Cellular Component: nucleoplasm; Golgi apparatus; nuclear body; cytoplasm; histone deacetylase complex; nucleus; cytosol Molecular Function:protein binding; NAD-dependent histone deacetylase activity (H3-K9 specific); protein kinase C binding; NAD-dependent histone deacetylase activity (H3-K14 specific); metal ion binding; histone deacetylase binding; protein deacetylase activity; NAD-dependent histone deacetylase activity (H4-K16 specific); histone deacetylase activity; transcription factor binding; transcription corepressor activity Biological Process: response to drug; regulation of skeletal muscle fiber development; establishment and/or maintenance of chromatin architecture; Notch signaling pathway; transcription, DNA-dependent; B cell activation; heart development; chromatin silencing; histone deacetylation; chromatin modification; negative regulation of transcription from RNA polymerase II promoter; response to cocaine; regulation of gene expression, epigenetic; osteoblast development; chromatin remodeling; cellular response to insulin stimulus; protein amino acid deacetylation; B cell differentiation; regulation of protein binding; positive regulation of transcription factor activity; negative regulation of osteoblast differentiation; positive regulation of transcription from RNA polymerase II promoter; inflammatory response; negative regulation of transcription, DNA-dependent; multicellular organismal response to stress |
| NCBI Summary: | Histones play a critical role in transcriptional regulation, cell cycle progression, and developmental events. Histone acetylation/deacetylation alters chromosome structure and affects transcription factor access to DNA. The protein encoded by this gene belongs to the class II histone deacetylase/acuc/apha family. It possesses histone deacetylase activity and represses transcription when tethered to a promoter. It coimmunoprecipitates only with HDAC3 family member and might form multicomplex proteins. It also interacts with myocyte enhancer factor-2 (MEF2) proteins, resulting in repression of MEF2-dependent genes. This gene is thought to be associated with colon cancer. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008] |
| UniProt Code: | Q9UQL6 |
| NCBI GenInfo Identifier: | 296434519 |
| NCBI Gene ID: | 10014 |
| NCBI Accession: | Q9UQL6.2 |
| UniProt Secondary Accession: | Q9UQL6,O60340, O60528, Q96DY4, C9JFV9, |
| UniProt Related Accession: | Q9UQL6 |
| Molecular Weight: | 1122 |
| NCBI Full Name: | Histone deacetylase 5 |
| NCBI Synonym Full Names: | histone deacetylase 5 |
| NCBI Official Symbol: | HDAC5 |
| NCBI Official Synonym Symbols: | HD5; NY-CO-9 |
| NCBI Protein Information: | histone deacetylase 5; antigen NY-CO-9 |
| UniProt Protein Name: | Histone deacetylase 5 |
| UniProt Synonym Protein Names: | Antigen NY-CO-9 |
| UniProt Gene Name: | HDAC5 |
| UniProt Entry Name: | HDAC5_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-HDAC5 (Phospho-Ser259) Antibody, Anti-HDAC5 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.)
