TIP60 (Phospho-Ser86) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01382
€599

Description

TIP60 (Phospho-Ser86) Cell-Based ELISA Kit

The TIP60 (Phospho-Ser86) Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor TIP60 (Phospho-Ser86) protein expression profile in cells. The kit can be used for measuring the relative amounts of TIP60 (Phospho-Ser86) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on TIP60 phosphorylation.

How does our TIP60 (Phospho-Ser86) Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of TIP60 (Phospho-Ser86) concentration is achieved by an indirect ELISA format. In essence, TIP60 (Phospho-Ser86) is captured by TIP60 (Phospho-Ser86) -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

TIP60 (Phospho-Ser86) Colorimetric Cell-Based ELISA Kit -Information

Product Name:TIP60 (Phospho-Ser86) Colorimetric Cell-Based ELISA Kit
Product Code/SKU:CBCAB01382
Description:The TIP60 (Phospho-Ser86) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect TIP60 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated TIP60 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on TIP60 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Reactivity:Human, Mouse
Assay Type:Cell-Based
Format:2 x 96-Well Microplates
Database Links:Gene ID: 10524, UniProt ID: Q92993, OMIM #: 601409, Unigene #: Hs.528299
NCBI Gene Symbol:KAT5
Sub Type:Phospho
Target Name:TIP60 (Phospho-Ser86)

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

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:

  • 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 ?l 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

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 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-TIP60 (Phospho-Ser86) Antibody, Anti-TIP60 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.)

TIP60 (Phospho-Ser86) - Protein Information

UniProt Protein Function:Tip60: a histone acetyl transferase (HAT) of the MYST family. Originally isolated as an HIV-1 TAT-interactive protein. Plays an important role in regulating chromatin remodeling, transcription and other nuclear processes by acetylating nuclear proteins. Plays a role in DNA repair and apoptosis. Three splice variants have been described.
UniProt Protein Details:

Protein type:Acetyltransferase; Nucleolus; Nuclear receptor co-regulator; EC 2.3.1.48

Chromosomal Location of Human Ortholog: 11q13

Cellular Component: NuA4 histone acetyltransferase complex; nucleolus; nucleoplasm; nucleus

Molecular Function:acetyltransferase activity; histone acetyltransferase activity; protein binding; transcription coactivator activity

Biological Process: DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator; DNA double-strand break processing; DNA replication; DNA synthesis during DNA repair; double-strand break repair; double-strand break repair via nonhomologous end joining; histone acetylation; negative regulation of interleukin-2 production; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; proteasomal ubiquitin-dependent protein catabolic process; response to ionizing radiation; strand displacement

NCBI Summary:The protein encoded by this gene belongs to the MYST family of histone acetyl transferases (HATs) and was originally isolated as an HIV-1 TAT-interactive protein. HATs play important roles in regulating chromatin remodeling, transcription and other nuclear processes by acetylating histone and nonhistone proteins. This protein is a histone acetylase that has a role in DNA repair and apoptosis and is thought to play an important role in signal transduction. Alternative splicing of this gene results in multiple transcript variants. [provided by RefSeq, Jul 2008]
UniProt Code:Q92993
NCBI GenInfo Identifier:30923328
NCBI Gene ID:10524
NCBI Accession:Q92993.2
UniProt Secondary Accession:Q92993,O95624, Q13430, Q17RW5, Q561W3, Q6GSE8, Q9BWK7 B4E3C7, C9JL99,
UniProt Related Accession:Q92993
Molecular Weight:56,292 Da
NCBI Full Name:Histone acetyltransferase KAT5
NCBI Synonym Full Names:lysine acetyltransferase 5
NCBI Official Symbol:KAT5  
NCBI Official Synonym Symbols:TIP; ESA1; PLIP; TIP60; cPLA2; HTATIP; ZC2HC5; HTATIP1  
NCBI Protein Information:histone acetyltransferase KAT5
UniProt Protein Name:Histone acetyltransferase KAT5
UniProt Synonym Protein Names:60 kDa Tat-interactive protein; Tip60; Histone acetyltransferase HTATIP; HIV-1 Tat interactive protein; Lysine acetyltransferase 5; cPLA(2)-interacting protein
Protein Family:3-ketoacyl-CoA thiolase
UniProt Gene Name:KAT5  
UniProt Entry Name:KAT5_HUMAN
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