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HDAC6 Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00041
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Autophagy
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

HDAC6 Colorimetric Cell-Based ELISA Kit

The HDAC6 Colorimetric Cell-Based ELISA Kit is a comprehensive and reliable assay kit designed for the accurate detection of HDAC6 levels in cell lysates and tissue homogenates. With high sensitivity and specificity, this kit provides precise measurements of HDAC6 activity, allowing for the quantification of this important enzyme in various biological samples.HDAC6, a histone deacetylase enzyme, plays a critical role in regulating cellular processes such as gene expression, protein folding, and cell signaling. Dysregulation of HDAC6 has been implicated in various diseases, including cancer, neurodegenerative disorders, and inflammatory conditions, making it a valuable target for therapeutic interventions.

This innovative ELISA kit offers researchers a powerful tool for studying the role of HDAC6 in disease pathogenesis and for evaluating potential therapeutic strategies targeting this enzyme. With its user-friendly protocol and reliable performance, the HDAC6 Colorimetric Cell-Based ELISA Kit is an essential addition to any laboratory conducting research in the field of epigenetics and molecular biology.

Product Name:HDAC6 Colorimetric Cell-Based ELISA
Product Code:CBCAB00041
ELISA Type:Cell-Based
Target:HDAC6
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The HDAC6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect HDAC6 protein expression profile in cells. The kit can be used for measuring the relative amounts of HDAC6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on HDAC6.

Qualitative determination of HDAC6 concentration is achieved by an indirect ELISA format. In essence, HDAC6 is captured by HDAC6-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: 10013, UniProt ID: Q9UBN7, OMIM: 300272, Unigene: Hs.6764
Gene Symbol:HDAC6
Sub Type:None
UniProt Protein Function:HDAC6: 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. Plays a central role in microtubule-dependent cell motility via deacetylation of tubulin. Interacts with CBFA2T3, HDAC11 and SIRT2. Interacts with F-actin. Interacts with BBIP10. Under proteasome impairment conditions, interacts with UBD via its histone deacetylase 1 and UBP-type zinc-finger regions. Interacts with CYLD. Interacts with ZMYND15. Belongs to the histone deacetylase family. HD type 2 subfamily.
UniProt Protein Details:

Protein type:Ubiquitin conjugating system; EC 3.5.1.98; Nuclear receptor co-regulator; Deacetylase

Chromosomal Location of Human Ortholog: Xp11.23

Cellular Component: microtubule; dendrite; histone deacetylase complex; leading edge; perikaryon; caveola; inclusion body; cytosol; nucleoplasm; dynein complex; microtubule associated complex; axon; perinuclear region of cytoplasm; cytoplasmic microtubule; cytoplasm; nucleus

Molecular Function:zinc ion binding; microtubule binding; histone deacetylase binding; beta-catenin binding; beta-tubulin binding; Hsp90 protein binding; misfolded protein binding; actin binding; NAD-dependent histone deacetylase activity (H3-K9 specific); protein binding; enzyme binding; NAD-dependent histone deacetylase activity (H3-K14 specific); tubulin deacetylase activity; ubiquitin protein ligase binding; NAD-dependent histone deacetylase activity (H4-K16 specific); histone deacetylase activity; polyubiquitin binding; tau protein binding; alpha-tubulin binding

Biological Process: negative regulation of proteolysis; response to misfolded protein; protein polyubiquitination; transcription, DNA-dependent; ubiquitin-dependent protein catabolic process via the multivesicular body pathway; positive regulation of signal transduction; regulation of fat cell differentiation; macroautophagy; negative regulation of microtubule depolymerization; response to toxin; organelle organization and biogenesis; misfolded or incompletely synthesized protein catabolic process; histone deacetylation; regulation of gene expression, epigenetic; response to organic substance; intracellular protein transport; protein complex disassembly; lysosome localization; protein amino acid deacetylation; negative regulation of oxidoreductase activity; regulation of receptor activity; negative regulation of transcription, DNA-dependent; negative regulation of protein complex disassembly

Disease: Chondrodysplasia With Platyspondyly, Distinctive Brachydactyly, Hydrocephaly, And Microphthalmia

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 II of the histone deacetylase/acuc/apha family. It contains an internal duplication of two catalytic domains which appear to function independently of each other. This protein possesses histone deacetylase activity and represses transcription. [provided by RefSeq, Jul 2008]
UniProt Code:Q9UBN7
NCBI GenInfo Identifier:205371758
NCBI Gene ID:10013
NCBI Accession:Q9UBN7.2
UniProt Secondary Accession:Q9UBN7,O94975, Q6NT75, Q7L3E5, Q96CY0,
UniProt Related Accession:Q9UBN7
Molecular Weight:114,361 Da
NCBI Full Name:Histone deacetylase 6
NCBI Synonym Full Names:histone deacetylase 6
NCBI Official Symbol:HDAC6  
NCBI Official Synonym Symbols:HD6; JM21; CPBHM; PPP1R90  
NCBI Protein Information:histone deacetylase 6; protein phosphatase 1, regulatory subunit 90
UniProt Protein Name:Histone deacetylase 6
UniProt Gene Name:HDAC6  
UniProt Entry Name:HDAC6_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-HDAC6 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.)

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