ASK1 Colorimetric Cell-Based ELISA Kit (CBCAB00274)

Product Name:	ASK1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00274
ELISA Type:	Cell-Based
Target:	ASK1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The ASK1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ASK1 protein expression profile in cells. The kit can be used for measuring the relative amounts of ASK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on ASK1.

Qualitative determination of ASK1 concentration is achieved by an indirect ELISA format. In essence, ASK1 is captured by ASK1-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: 4217, UniProt ID: Q99683, OMIM: 602448, Unigene: Hs.186486
Gene Symbol:	MAP3K5
Sub Type:	None

UniProt Protein Function:	ASK1: a MAP kinase kinase kinase of the STE11 family, apoptosis signal-regulating kinase 1 (ASK1) plays essential roles in stress-induced apoptosis. Activated by various stressors, including oxidative stress, endoplasmic reticulum stress, and calcium overload, as well as by receptor-mediated inflammatory signals, such as the tumor necrosis factor (TNF) and lipopolysaccharide (LPS). In turn, ASK1 phosphorylates and activates two different subgroups of MAP kinase kinases, MKK4/SEK1 and MKK3/MKK6, which in turn activate stress-activated protein kinases (JNKs) and p38 subgroups of MAP kinases, respectively. Overexpression activates JNK and p38 and induces apoptosis in several cell types through signals involving the mitochondrial cell death pathway. Embryonic fibroblasts or primary neurons derived from ASK1-/- mice are resistant to stress-induced JNK and p38 activation as well as cell death. Oxidative stress induces the activation of ASK1, which correlate with ASK1 activity and ASK1-dependent apoptosis. Required for the innate immune response, which is essential for host defense against a wide range of pathogens.
UniProt Protein Details:	Protein type:EC 2.7.11.25; Kinase, protein; Protein kinase, STE; Protein kinase, Ser/Thr (non-receptor); STE group; STE11 family Chromosomal Location of Human Ortholog: 6q23.3 Cellular Component: cytosol Molecular Function:ATP binding; magnesium ion binding; MAP kinase kinase kinase activity; protein binding; protein homodimerization activity; protein kinase activity; protein phosphatase binding; protein serine/threonine kinase activity Biological Process: activation of JNK activity; activation of MAPKK activity; induction of apoptosis by oxidative stress; JNK cascade; MAPKKK cascade; positive regulation of apoptosis; positive regulation of caspase activity; positive regulation of JNK activity; positive regulation of JNK cascade; protein amino acid phosphorylation
NCBI Summary:	Mitogen-activated protein kinase (MAPK) signaling cascades include MAPK or extracellular signal-regulated kinase (ERK), MAPK kinase (MKK or MEK), and MAPK kinase kinase (MAPKKK or MEKK). MAPKK kinase/MEKK phosphorylates and activates its downstream protein kinase, MAPK kinase/MEK, which in turn activates MAPK. The kinases of these signaling cascades are highly conserved, and homologs exist in yeast, Drosophila, and mammalian cells. MAPKKK5 contains 1,374 amino acids with all 11 kinase subdomains. Northern blot analysis shows that MAPKKK5 transcript is abundantly expressed in human heart and pancreas. The MAPKKK5 protein phosphorylates and activates MKK4 (aliases SERK1, MAPKK4) in vitro, and activates c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) during transient expression in COS and 293 cells; MAPKKK5 does not activate MAPK/ERK. [provided by RefSeq, Jul 2008]
UniProt Code:	Q99683
NCBI GenInfo Identifier:	6685617
NCBI Gene ID:	4217
NCBI Accession:	Q99683.1
UniProt Secondary Accession:	Q99683,Q5THN3, Q99461, A6NIA0, B4DGB2,
UniProt Related Accession:	Q99683
Molecular Weight:	69,839 Da
NCBI Full Name:	Mitogen-activated protein kinase kinase kinase 5
NCBI Synonym Full Names:	mitogen-activated protein kinase kinase kinase 5
NCBI Official Symbol:	MAP3K5
NCBI Official Synonym Symbols:	ASK1; MEKK5; MAPKKK5
NCBI Protein Information:	mitogen-activated protein kinase kinase kinase 5
UniProt Protein Name:	Mitogen-activated protein kinase kinase kinase 5
UniProt Synonym Protein Names:	Apoptosis signal-regulating kinase 1; ASK-1; MAPK/ERK kinase kinase 5; MEK kinase 5; MEKK 5
Protein Family:	DASH complex
UniProt Gene Name:	MAP3K5

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-ASK1 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.)