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ALK (Phospho-Tyr1604)Colorimetric Cell-Based ELISA Kit (CBCAB01337)

SKU:
CBCAB01337
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Detection Method:
Colorimetric
€960
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Description

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ALK (Phospho-Tyr1604)Colorimetric Cell-Based ELISA Kit

The ALK (Anaplastic Lymphoma Kinase) Phospho-Tyr1604 Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the precise measurement of ALK phosphorylation at tyrosine 1604 in cell lysates. This kit offers high sensitivity and specificity, ensuring accurate and reproducible results for researchers studying kinase signaling pathways and exploring potential therapeutic targets.ALK is a receptor tyrosine kinase that plays a critical role in cell growth, survival, and differentiation. Dysregulation of ALK signaling has been implicated in various cancers, including anaplastic large cell lymphoma and non-small cell lung cancer.

Monitoring ALK phosphorylation at specific sites, such as Tyr1604, can provide valuable insights into the activation status of this kinase and its downstream signaling pathways.The ALK Phospho-Tyr1604 Colorimetric Cell-Based ELISA Kit is easy to use and allows for high-throughput screening of multiple samples simultaneously. With its reliable performance and broad applicability, this kit is an essential tool for studying ALK signaling dynamics and identifying potential therapeutic targets in cancer research and drug development.

Product Name:ALK (Phospho-Tyr1604) Colorimetric Cell-Based ELISA
Product Code:CBCAB01337
ELISA Type:Cell-Based
Target:ALK (Phospho-Tyr1604)
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The ALK (Phospho-Tyr1604) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect ALK protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated ALK in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on ALK phosphorylation.

Qualitative determination of ALK (Phospho-Tyr1604) concentration is achieved by an indirect ELISA format. In essence, ALK (Phospho-Tyr1604) is captured by ALK (Phospho-Tyr1604)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 238, UniProt ID: Q9UM73, OMIM: 105590, Unigene: Hs.654469
Gene Symbol:ALK
Sub Type:Phospho
UniProt Protein Function:ALK: a tyrosine kinase of the ALK family. Plays an important role in the development of the brain and exerts its effects on specific neurons in the nervous system. Translocated and expressed as a fusion protein in anaplastic lymphoma. About one third of large-cell lymphomas are caused by a t(2;5)(p23;q35) translocation that fuses ALK to nucleophosmin (NPM1A). Other cases caused by fusions of ALK to moesin, non-muscle myosin heavy chain 9, clathrin heavy chain and other genes. Several fusions also seen in inflammatory myofibroblastic tumors, and expression has been briefly noted in a range of tumors
UniProt Protein Details:

Protein type:EC 2.7.10.1; Protein kinase, TK; Membrane protein, integral; Kinase, protein; Protein kinase, tyrosine (receptor); Oncoprotein; TK group; Alk family

Chromosomal Location of Human Ortholog: 2p23

Cellular Component: protein complex; integral to plasma membrane

Molecular Function:NF-kappaB-inducing kinase activity; protein binding; protein-tyrosine kinase activity; transmembrane receptor protein tyrosine kinase activity; ATP binding

Biological Process: regulation of apoptosis; cell proliferation; peptidyl-tyrosine phosphorylation; activation of MAPK activity; protein amino acid autophosphorylation; neuron development; signal transduction; phosphorylation; transmembrane receptor protein tyrosine kinase signaling pathway; activation of NF-kappaB transcription factor

Disease: Neuroblastoma, Susceptibility To, 3

NCBI Summary:This gene encodes a receptor tyrosine kinase, which belongs to the insulin receptor superfamily. This protein comprises an extracellular domain, an hydrophobic stretch corresponding to a single pass transmembrane region, and an intracellular kinase domain. It plays an important role in the development of the brain and exerts its effects on specific neurons in the nervous system. This gene has been found to be rearranged, mutated, or amplified in a series of tumours including anaplastic large cell lymphomas, neuroblastoma, and non-small cell lung cancer. The chromosomal rearrangements are the most common genetic alterations in this gene, which result in creation of multiple fusion genes in tumourigenesis, including ALK (chromosome 2)/EML4 (chromosome 2), ALK/RANBP2 (chromosome 2), ALK/ATIC (chromosome 2), ALK/TFG (chromosome 3), ALK/NPM1 (chromosome 5), ALK/SQSTM1 (chromosome 5), ALK/KIF5B (chromosome 10), ALK/CLTC (chromosome 17), ALK/TPM4 (chromosome 19), and ALK/MSN (chromosome X).[provided by RefSeq, Jan 2011]
UniProt Code:Q9UM73
NCBI GenInfo Identifier:296439447
NCBI Gene ID:238
NCBI Accession:Q9UM73.3
UniProt Secondary Accession:Q9UM73,Q4ZFX9, Q53QQ6, Q53RZ4, Q59FI3, Q9Y4K6,
UniProt Related Accession:Q9UM73
Molecular Weight:176,442 Da
NCBI Full Name:ALK tyrosine kinase receptor
NCBI Synonym Full Names:anaplastic lymphoma receptor tyrosine kinase
NCBI Official Symbol:ALK  
NCBI Official Synonym Symbols:CD246; NBLST3  
NCBI Protein Information:ALK tyrosine kinase receptor; CD246 antigen; mutant anaplastic lymphoma kinase
UniProt Protein Name:ALK tyrosine kinase receptor
UniProt Synonym Protein Names:Anaplastic lymphoma kinase
Protein Family:ALK tyrosine kinase receptor
UniProt Gene Name:ALK  
UniProt Entry Name:ALK_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-ALK (Phospho-Tyr1604) Antibody, Anti-ALK 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.)

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