MTA1 Colorimetric Cell-Based ELISA (CBCAB01082)

Product Name:	MTA1 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01082
ELISA Type:	Cell-Based
Target:	MTA1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MTA1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MTA1 protein expression profile in cells. The kit can be used for measuring the relative amounts of MTA1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MTA1.

Qualitative determination of MTA1 concentration is achieved by an indirect ELISA format. In essence, MTA1 is captured by MTA1-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: 9112, UniProt ID: Q13330, OMIM: 603526, Unigene: Hs.525629
Gene Symbol:	MTA1
Sub Type:	None

UniProt Protein Function:	MTA1: metastasis-associated protein. A component of the NuRD ATP-dependent chromatin remodeling and histone deacetylase complex. Interacts with HDAC1. Expressed at high levels in metastatic cells. Two alternatively spliced isoforms have been described. The long isoform is a co-repressor of estrogen receptor (ER). The short isoform binds to ER and sequesters it in the cytoplasm and enhances non-genomic responses of ER.
UniProt Protein Details:	Protein type:Nuclear receptor co-regulator; Transcription, coactivator/corepressor; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 14q32.3 Cellular Component: cytoplasm; intracellular membrane-bound organelle; microtubule; nuclear envelope; nucleoplasm; nucleus; NuRD complex Molecular Function:chromatin binding; protein binding; transcription coactivator activity; transcription corepressor activity; transcription factor activity; zinc ion binding Biological Process: cellular protein metabolic process; circadian regulation of gene expression; double-strand break repair; entrainment of circadian clock by photoperiod; establishment and/or maintenance of chromatin architecture; locomotor rhythm; post-translational protein modification; proteasomal ubiquitin-dependent protein catabolic process; protein sumoylation; regulation of gene expression, epigenetic; regulation of inflammatory response; regulation of transcription, DNA-dependent; response to ionizing radiation; response to lipopolysaccharide; signal transduction; transcription, DNA-dependent
NCBI Summary:	This gene encodes a protein that was identified in a screen for genes expressed in metastatic cells, specifically, mammary adenocarcinoma cell lines. Expression of this gene has been correlated with the metastatic potential of at least two types of carcinomas although it is also expressed in many normal tissues. The role it plays in metastasis is unclear. It was initially thought to be the 70kD component of a nucleosome remodeling deacetylase complex, NuRD, but it is more likely that this component is a different but very similar protein. These two proteins are so closely related, though, that they share the same types of domains. These domains include two DNA binding domains, a dimerization domain, and a domain commonly found in proteins that methylate DNA. The profile and activity of this gene product suggest that it is involved in regulating transcription and that this may be accomplished by chromatin remodeling. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Feb 2011]
UniProt Code:	Q13330
NCBI GenInfo Identifier:	259016275
NCBI Gene ID:	9112
NCBI Accession:	Q13330.2
UniProt Secondary Accession:	Q13330,Q86SW2, Q8NFI8, Q96GI8, A5PLK4,
UniProt Related Accession:	Q13330
Molecular Weight:	79,192 Da
NCBI Full Name:	Metastasis-associated protein MTA1
NCBI Synonym Full Names:	metastasis associated 1
NCBI Official Symbol:	MTA1
NCBI Protein Information:	metastasis-associated protein MTA1
UniProt Protein Name:	Metastasis-associated protein MTA1
Protein Family:	Mating type protein
UniProt Gene Name:	MTA1
UniProt Entry Name:	MTA1_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-MTA1 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.)