STK39 Colorimetric Cell-Based ELISA Kit (CBCAB00874)

Product Name:	STK39 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00874
ELISA Type:	Cell-Based
Target:	STK39
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The STK39 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect STK39 protein expression profile in cells. The kit can be used for measuring the relative amounts of STK39 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on STK39.

Qualitative determination of STK39 concentration is achieved by an indirect ELISA format. In essence, STK39 is captured by STK39-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: 27347, UniProt ID: Q9UEW8, OMIM: 607648, Unigene: Hs.276271
Gene Symbol:	STK39
Sub Type:	None

UniProt Protein Function:	STLK3: May act as a mediator of stress-activated signals. The phosphorylated form forms a complex with WNK2. Predominantly expressed in brain and pancreas followed by heart, lung, kidney, skeletal muscle, liver, placenta and testis. Belongs to the protein kinase superfamily. STE Ser/Thr protein kinase family. STE20 subfamily.
UniProt Protein Details:	Protein type:Kinase, protein; EC 2.7.11.1; Protein kinase, STE; Protein kinase, Ser/Thr (non-receptor); STE group; STE20 family; FRAY subfamily Chromosomal Location of Human Ortholog: 2q24.3 Cellular Component: apical plasma membrane; basolateral plasma membrane; cytoplasm; cytoskeleton; nucleus Molecular Function:ATP binding; protein binding; protein kinase binding; protein serine/threonine kinase activity; receptor signaling protein serine/threonine kinase activity Biological Process: activation of protein kinase activity; negative regulation of protein amino acid phosphorylation; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; positive regulation of potassium ion transport; protein amino acid phosphorylation; regulation of apoptosis; regulation of blood pressure; regulation of inflammatory response; regulation of mitotic cell cycle; response to stress; stress-activated protein kinase signaling pathway
NCBI Summary:	This gene encodes a serine/threonine kinase that is thought to function in the cellular stress response pathway. The kinase is activated in response to hypotonic stress, leading to phosphorylation of several cation-chloride-coupled cotransporters. The catalytically active kinase specifically activates the p38 MAP kinase pathway, and its interaction with p38 decreases upon cellular stress, suggesting that this kinase may serve as an intermediate in the response to cellular stress. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9UEW8
NCBI GenInfo Identifier:	317373508
NCBI Gene ID:	27347
NCBI Accession:	Q9UEW8.3
UniProt Secondary Accession:	Q9UEW8,O14774, Q53S90, Q53SL7, Q53SS1, Q9UER4, X5D9C8
UniProt Related Accession:	Q9UEW8
Molecular Weight:	57,937 Da
NCBI Full Name:	STE20/SPS1-related proline-alanine-rich protein kinase
NCBI Synonym Full Names:	serine/threonine kinase 39
NCBI Official Symbol:	STK39
NCBI Official Synonym Symbols:	DCHT; PASK; SPAK
NCBI Protein Information:	STE20/SPS1-related proline-alanine-rich protein kinase
UniProt Protein Name:	STE20/SPS1-related proline-alanine-rich protein kinase
UniProt Synonym Protein Names:	DCHT; Serine/threonine-protein kinase 39
Protein Family:	STE20/SPS1-related proline-alanine-rich protein kinase
UniProt Gene Name:	STK39
UniProt Entry Name:	STK39_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-STK39 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.)