HDAC2 (Phospho-Ser394) Colorimetric Cell-Based ELISA Kit
- SKU:
- CBCAB01538
- Product Type:
- ELISA Kit
- ELISA Type:
- Cell Based Phospho Specific
- Research Area:
- Cell Biology
- Reactivity:
- Human
- Reactivity:
- Mouse
- Reactivity:
- Rat
- Detection Method:
- Colorimetric
Description
| Product Name: | HDAC2 (Phospho-Ser394) Colorimetric Cell-Based ELISA |
| Product Code: | CBCAB01538 |
| ELISA Type: | Cell-Based |
| Target: | HDAC2 (Phospho-Ser394) |
| Reactivity: | Human, Mouse, Rat |
| Dynamic Range: | > 5000 Cells |
| Detection Method: | Colorimetric 450 nm |
| Format: | 2 x 96-Well Microplates |
The HDAC2 (Phospho-Ser394) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HDAC2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HDAC2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on HDAC2 phosphorylation.
Qualitative determination of HDAC2 (Phospho-Ser394) concentration is achieved by an indirect ELISA format. In essence, HDAC2 (Phospho-Ser394) is captured by HDAC2 (Phospho-Ser394)-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: 3066, UniProt ID: Q92769, OMIM: 605164, Unigene: Hs.3352 |
| Gene Symbol: | HDAC2 |
| Sub Type: | Phospho |
| UniProt Protein Function: | HDAC2: a transcriptional regulator of the histone deacetylase family, subfamily 1. Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation plays a role in epigenetic repression and transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes. |
| UniProt Protein Details: | Protein type:EC 3.5.1.98; Transcription, coactivator/corepressor; Deacetylase; Nuclear receptor co-regulator Chromosomal Location of Human Ortholog: 6q21 Cellular Component: nucleoplasm; heterochromatin; transcription factor complex; protein complex; Sin3 complex; ESC/E(Z) complex; cytoplasm; nuclear chromatin; replication fork; NuRD complex; nucleus Molecular Function:deacetylase activity; nucleosomal DNA binding; protein deacetylase activity; transcription factor binding; NAD-dependent histone deacetylase activity (H3-K9 specific); protein binding; enzyme binding; NAD-dependent histone deacetylase activity (H3-K14 specific); sequence-specific DNA binding; heat shock protein binding; chromatin binding; NAD-dependent histone deacetylase activity (H4-K16 specific); histone deacetylase activity; transcription factor activity Biological Process: establishment and/or maintenance of chromatin architecture; nerve growth factor receptor signaling pathway; positive regulation of proteolysis; positive regulation of transcription, DNA-dependent; positive regulation of collagen biosynthetic process; negative regulation of transcription from RNA polymerase II promoter; regulation of gene expression, epigenetic; negative regulation of cell cycle; cardiac muscle cell development; negative regulation of cardiac muscle cell proliferation; negative regulation of gene expression, epigenetic; positive regulation of cell proliferation; dendrite development; circadian regulation of gene expression; positive regulation of oligodendrocyte differentiation; response to drug; transcription, DNA-dependent; negative regulation of transcription factor activity; hippocampus development; histone deacetylation; regulation of protein kinase B signaling cascade; response to cocaine; odontogenesis of dentine-containing teeth; chromatin remodeling; maintenance of chromatin silencing; negative regulation of MHC class II biosynthetic process; ATP-dependent chromatin remodeling; epidermal cell differentiation; positive regulation of transcription factor activity; gene expression; positive regulation of transcription from RNA polymerase II promoter; embryonic digit morphogenesis; blood coagulation; negative regulation of transcription, DNA-dependent; negative regulation of apoptosis |
| NCBI Summary: | This gene product belongs to the histone deacetylase family. Histone deacetylases act via the formation of large multiprotein complexes, and are responsible for the deacetylation of lysine residues at the N-terminal regions of core histones (H2A, H2B, H3 and H4). This protein forms transcriptional repressor complexes by associating with many different proteins, including YY1, a mammalian zinc-finger transcription factor. Thus, it plays an important role in transcriptional regulation, cell cycle progression and developmental events. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2010] |
| UniProt Code: | Q92769 |
| NCBI GenInfo Identifier: | 68068066 |
| NCBI Gene ID: | 3066 |
| NCBI Accession: | Q92769.2 |
| UniProt Secondary Accession: | Q92769,Q5SRI8, Q5SZ86, Q8NEH4, B3KRS5, B4DL58, E1P561 |
| UniProt Related Accession: | Q92769 |
| Molecular Weight: | 488 |
| NCBI Full Name: | Histone deacetylase 2 |
| NCBI Synonym Full Names: | histone deacetylase 2 |
| NCBI Official Symbol: | HDAC2 |
| NCBI Official Synonym Symbols: | HD2; RPD3; YAF1 |
| NCBI Protein Information: | histone deacetylase 2; YY1-associated factor 1; transcriptional regulator homolog RPD3 |
| UniProt Protein Name: | Histone deacetylase 2 |
| UniProt Gene Name: | HDAC2 |
| UniProt Entry Name: | HDAC2_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-HDAC2 (Phospho-Ser394) Antibody, Anti-HDAC2 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.)
