DUSP4 Colorimetric Cell-Based ELISA (CBCAB01078)

Product Name:	DUSP4 Colorimetric Cell-Based ELISA
Product Code:	CBCAB01078
ELISA Type:	Cell-Based
Target:	DUSP4
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The DUSP4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect DUSP4 protein expression profile in cells. The kit can be used for measuring the relative amounts of DUSP4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on DUSP4.

Qualitative determination of DUSP4 concentration is achieved by an indirect ELISA format. In essence, DUSP4 is captured by DUSP4-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: 1846, UniProt ID: Q13115, OMIM: 602747, Unigene: Hs.417962
Gene Symbol:	DUS4
Sub Type:	None

UniProt Protein Function:	MKP-2: a non-receptor, dual-specificity phosphoprotein phosphatase (DUSP). Different members of the DUSP family show distinct substrate specificities for MAPKs, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. DUSP4 inactivates ERK1, ERK2 and JNK, is expressed in a variety of tissues, and is localized in the nucleus. Induced by hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) in human endothelial cells. Two alternatively spliced isoforms have been described. In addition, multiple polyadenylation sites have been reported. Contains 1 rhodanese domain.
UniProt Protein Details:	Protein type:Protein phosphatase, dual-specificity; EC 3.1.3.48; EC 3.1.3.16 Chromosomal Location of Human Ortholog: 8p12-p11 Cellular Component: nucleoplasm; nucleus Molecular Function:MAP kinase tyrosine/serine/threonine phosphatase activity; protein tyrosine phosphatase activity; protein tyrosine/threonine phosphatase activity Biological Process: endoderm formation; inactivation of MAPK activity; innate immune response; MAPKKK cascade; MyD88-dependent toll-like receptor signaling pathway; MyD88-independent toll-like receptor signaling pathway; nerve growth factor receptor signaling pathway; protein amino acid dephosphorylation; stress-activated MAPK cascade; toll-like receptor 10 signaling pathway; toll-like receptor 2 signaling pathway; toll-like receptor 3 signaling pathway; toll-like receptor 4 signaling pathway; toll-like receptor 5 signaling pathway; toll-like receptor 9 signaling pathway; toll-like receptor signaling pathway
NCBI Summary:	The protein encoded by this gene is a member of the dual specificity protein phosphatase subfamily. These phosphatases inactivate their target kinases by dephosphorylating both the phosphoserine/threonine and phosphotyrosine residues. They negatively regulate members of the mitogen-activated protein (MAP) kinase superfamily (MAPK/ERK, SAPK/JNK, p38), which are associated with cellular proliferation and differentiation. Different members of the family of dual specificity phosphatases show distinct substrate specificities for various MAP kinases, different tissue distribution and subcellular localization, and different modes of inducibility of their expression by extracellular stimuli. This gene product inactivates ERK1, ERK2 and JNK, is expressed in a variety of tissues, and is localized in the nucleus. Two alternatively spliced transcript variants, encoding distinct isoforms, have been observed for this gene. In addition, multiple polyadenylation sites have been reported. [provided by RefSeq, Jul 2008]
UniProt Code:	Q13115
NCBI GenInfo Identifier:	2499745
NCBI Gene ID:	1846
NCBI Accession:	Q13115.1
UniProt Secondary Accession:	Q13115,Q13524, B2RBU5, D3DSU4, G5E930,
UniProt Related Accession:	Q13115
Molecular Weight:	32,993 Da
NCBI Full Name:	Dual specificity protein phosphatase 4
NCBI Synonym Full Names:	dual specificity phosphatase 4
NCBI Official Symbol:	DUSP4
NCBI Official Synonym Symbols:	TYP; HVH2; MKP2; MKP-2
NCBI Protein Information:	dual specificity protein phosphatase 4
UniProt Protein Name:	Dual specificity protein phosphatase 4
UniProt Synonym Protein Names:	Dual specificity protein phosphatase hVH2; Mitogen-activated protein kinase phosphatase 2; MAP kinase phosphatase 2; MKP-2
Protein Family:	Dual specificity protein phosphatase
UniProt Gene Name:	DUSP4
UniProt Entry Name:	DUS4_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-DUSP4 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.)