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HDAC5 (Phospho-Ser259) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00023
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Fluorometric
$719
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Description

Technical Manual

HDAC5 (Phospho-Ser259) Cell-Based ELISA Kit

The HDAC5 (Phospho-Ser259) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor HDAC5 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.

How does our HDAC5 (Phospho-Ser259) Fluorometric Cell-Based ELISA Kit work?

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 (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2. An antibody against the nonphosphorylated counterpart of HDAC5 (Phospho-Ser259) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated HDAC5 can be used to normalize the RFU value for phosphorylated HDAC5.

HDAC5 (Phospho-Ser259) Fluorometric Cell-Based ELISA Kit -Information

Product Name:HDAC5 (Phospho-Ser259) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00023
Description:The HDAC5 (Phospho-Ser259) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor HDAC5 (Phospho-Ser259) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated HDAC5 (Phospho-Ser259) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on HDAC5/HDAC9 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 10014/9734, UniProt ID: Q9UQL6, OMIM #: 605315, Unigene #: Hs.438782
Format:Two 96-Well Plates
NCBI Gene Symbol:HDAC5/HDAC9
Sub Type:Phospho
Target Name:Phospho-HDAC5 (Ser259)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components

Quantity

96-Well Black Cell CultureClear-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 Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
  • Micropipettes capable of measuring volumes from 1 µl to 1 ml
  • Deionized or sterile water (ddH2O)
  • 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)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.Dispense 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 3 minutes each time.
11.Add 50 µl of Primary Antibody Mixture P to corresponding wells for HDAC5 (Phospho-Ser259) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total HDAC5 detection. Cover the plate 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 3 minutes each time.
13.Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

HDAC5 (Phospho-Ser259) - Protein Information

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

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