Description
Human Histone deacetylase 8 (HDAC8) ELISA Kit
The Human Histone Deacetylase 8 (HDAC8) ELISA Kit is a powerful tool for the quantitative measurement of HDAC8 levels in human samples such as serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.HDAC8 is an important enzyme involved in regulating gene expression by modifying histone proteins. Dysregulation of HDAC8 has been linked to various diseases, including cancer, neurodevelopmental disorders, and cardiovascular diseases.
Therefore, monitoring HDAC8 levels can provide valuable insights into disease progression and potential treatment options.Overall, the Human HDAC8 ELISA Kit is a valuable resource for researchers looking to investigate the role of HDAC8 in health and disease, offering a reliable and efficient means of quantifying HDAC8 levels in human samples.
| Product Name: | Human Histone deacetylase 8 (HDAC8) ELISA Kit |
| SKU: | HUEB1112 |
| Size: | 96T |
| Target: | Human Histone deacetylase 8 (HDAC8) |
| Synonyms: | CDA07, HD8, HDACL1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.625-40ng/mL |
| Sensitivity: | 0.32ng/mL |
| Intra CV: | 5.4% | ||||||||||||||||||||
| Inter CV: | 8.3% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes. Also involved in the deacetylation of cohesin complex protein SMC3 regulating release of cohesin complexes from chromatin. May play a role in smooth muscle cell contractility. |
| Uniprot: | Q9BY41 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Histone deacetylase 8 |
| Sub Unit: | Interacts with PEPB2-MYH11, a fusion protein consisting of the 165 N-terminal residues of CBF-beta (PEPB2) with the tail region of MYH11 produced by the inversion Inv(16)(p13q22), a translocation associated with acute myeloid leukemia of M4EO subtype. The PEPB2-MYH1 fusion protein also interacts with RUNX1, a well known transcriptional regulator, suggesting that the interaction with HDAC8 may participate in the conversion of RUNX1 into a constitutive transcriptional repressor. Interacts with CBFA2T3. Interacts with phosphorylated SMG5/EST1B; this interaction protects SMG5 from ubiquitin-mediated degradation. Associates with alpha-SMA (smooth muscle alpha-actin). |
| Research Area: | Cancer |
| Subcellular Location: | Nucleus Cytoplasm Excluded from the nucleoli. Found in the cytoplasm of cells showing smooth muscle differentiation. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | HDAC8: Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes. May play a role in smooth muscle cell contractility. Belongs to the histone deacetylase family. HD type 1 subfamily. 5 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:EC 3.5.1.98; Hydrolase Chromosomal Location of Human Ortholog: Xq13 Cellular Component: nucleoplasm; nuclear chromosome; histone deacetylase complex; cytoplasm; plasma membrane; cytosol; nucleus Molecular Function:NAD-dependent histone deacetylase activity (H3-K9 specific); NAD-dependent histone deacetylase activity (H3-K14 specific); metal ion binding; NAD-dependent histone deacetylase activity (H4-K16 specific); histone deacetylase activity; transcription factor binding Biological Process: chromatin assembly or disassembly; establishment and/or maintenance of chromatin architecture; transcription, DNA-dependent; sister chromatid cohesion; mitotic cell cycle; chromatin modification; negative regulation of transcription from RNA polymerase II promoter Disease: Wilson-turner X-linked Mental Retardation Syndrome |
| 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 class I of the histone deacetylase family. It catalyzes the deacetylation of lysine residues in the histone N-terminal tails and represses transcription in large multiprotein complexes with transcriptional co-repressors. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Oct 2009] |
| UniProt Code: | Q9BY41 |
| NCBI GenInfo Identifier: | 29839394 |
| NCBI Gene ID: | 55869 |
| NCBI Accession: | Q9BY41.2 |
| UniProt Secondary Accession: | Q9BY41,Q86VC8, Q9NP76, Q9NYH4, A6ND12, A6ND61, A6NET3 A6NJR3, A8MQ62, B4DKN0, B4DV22, |
| UniProt Related Accession: | Q9BY41 |
| Molecular Weight: | 377 |
| NCBI Full Name: | Histone deacetylase 8 |
| NCBI Synonym Full Names: | histone deacetylase 8 |
| NCBI Official Symbol: | HDAC8 |
| NCBI Official Synonym Symbols: | HD8; WTS; RPD3; CDA07; CDLS5; MRXS6; HDACL1 |
| NCBI Protein Information: | histone deacetylase 8; histone deacetylase-like 1 |
| UniProt Protein Name: | Histone deacetylase 8 |
| UniProt Gene Name: | HDAC8 |
| UniProt Entry Name: | HDAC8_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
