JAK1 (Phospho-Tyr1022)Colorimetric Cell-Based ELISA Kit (CBCAB00470)

Product Name:	JAK1 (Phospho-Tyr1022)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00470
ELISA Type:	Cell-Based
Target:	JAK1 (Phospho-Tyr1022)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The JAK1 (Phospho-Tyr1022) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect JAK1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated JAK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on JAK1 phosphorylation.

Qualitative determination of JAK1 (Phospho-Tyr1022) concentration is achieved by an indirect ELISA format. In essence, JAK1 (Phospho-Tyr1022) is captured by JAK1 (Phospho-Tyr1022)-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: 3716, UniProt ID: P23458, OMIM: 147795, Unigene: Hs.207538
Gene Symbol:	JAK1
Sub Type:	Phospho

UniProt Protein Function:	JAK1: a widely expressed non-receptor tyrosine-kinase involved in the interferon-alpha/beta and -gamma signal transduction pathways. Couples cytokine ligand binding to tyrosine phosphorylation of various known signaling proteins and of a unique family of transcription factors termed the signal transducers and activators of transcription, or STATs. A selective Jak1 inhibitor induces apoptosis in NRP-154 prostate cancer cell line. A single mutation seen in tyrosine kinome screen of colon cancers. Activated in B cell lines from patients with post-transplant lymphoproliferative disorder, and in a mouse Lck-driven T cell lymphoma. Inhibitor: Piceatannol.
UniProt Protein Details:	Protein type:Kinase, protein; EC 2.7.10.2; Protein kinase, TK; Protein kinase, tyrosine (non-receptor); TK group; JakA family Chromosomal Location of Human Ortholog: 1p32.3-p31.3 Cellular Component: cytoplasm; cytoskeleton; cytosol; endomembrane system; extrinsic to internal side of plasma membrane; focal adhesion; nucleus Molecular Function:ATP binding; CCR5 chemokine receptor binding; growth hormone receptor binding; metal ion binding; non-membrane spanning protein tyrosine kinase activity; protein binding; protein phosphatase binding; protein-tyrosine kinase activity; Ras guanyl-nucleotide exchange factor activity; receptor binding; ubiquitin protein ligase binding Biological Process: cell differentiation; cell migration; innate immune response; MAPKKK cascade; positive regulation of GTPase activity; protein amino acid phosphorylation; regulation of cell proliferation; response to antibiotic; transmembrane receptor protein tyrosine kinase signaling pathway
NCBI Summary:	This gene encodes a membrane protein that is a member of a class of protein-tyrosine kinases (PTK) characterized by the presence of a second phosphotransferase-related domain immediately N-terminal to the PTK domain. The encoded kinase phosphorylates STAT proteins (signal transducers and activators of transcription) and plays a key role in interferon-alpha/beta and interferon-gamma signal transduction. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Mar 2016]
UniProt Code:	P23458
NCBI GenInfo Identifier:	215274013
NCBI Gene ID:	3716
NCBI Accession:	P23458.2
UniProt Secondary Accession:	P23458,Q59GQ2, Q9UD26,
UniProt Related Accession:	P23458
Molecular Weight:
NCBI Full Name:	Tyrosine-protein kinase JAK1
NCBI Synonym Full Names:	Janus kinase 1
NCBI Official Symbol:	JAK1
NCBI Official Synonym Symbols:	JTK3; JAK1A; JAK1B
NCBI Protein Information:	tyrosine-protein kinase JAK1
UniProt Protein Name:	Tyrosine-protein kinase JAK1
UniProt Synonym Protein Names:	Janus kinase 1; JAK-1
Protein Family:	Tyrosine-protein kinase
UniProt Gene Name:	JAK1
UniProt Entry Name:	JAK1_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-JAK1 (Phospho-Tyr1022) Antibody, Anti-JAK1 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.)