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Csk (Phospho-Ser364) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00228
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Immunology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
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Description

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Csk (Phospho-Ser364)Colorimetric Cell-Based ELISA Kit

The CSK (C-terminal Src kinase) Phospho-Ser364 Colorimetric Cell-Based ELISA Kit is an innovative assay designed to accurately measure the phosphorylation levels of CSK at serine 364 in cell lysates. This kit offers high sensitivity and specificity, providing precise and reproducible results for researchers studying signal transduction pathways involving CSK.CSK is a tyrosine kinase that plays a crucial role in regulating cellular processes such as cell growth, differentiation, and apoptosis. Phosphorylation of CSK at serine 364 is known to modulate its kinase activity and interaction with other signaling molecules, making it a key player in various cellular signaling pathways.

The CSK Phospho-Ser364 Colorimetric Cell-Based ELISA Kit is essential for understanding the molecular mechanisms underlying key cellular processes and for identifying potential therapeutic targets in diseases such as cancer, autoimmune disorders, and inflammatory conditions. With its user-friendly protocol and reliable performance, this kit is a valuable tool for researchers looking to advance their understanding of cellular signaling networks.

Product Name:Csk (Phospho-Ser364) Colorimetric Cell-Based ELISA
Product Code:CBCAB00228
ELISA Type:Cell-Based
Target:Csk (Phospho-Ser364)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Csk (Phospho-Ser364) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Csk protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Csk in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Csk phosphorylation.

Qualitative determination of Csk (Phospho-Ser364) concentration is achieved by an indirect ELISA format. In essence, Csk (Phospho-Ser364) is captured by Csk (Phospho-Ser364)-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: 1445, UniProt ID: P41240, OMIM: 124095, Unigene: Hs.77793
Gene Symbol:CSK
Sub Type:Phospho
UniProt Protein Function:CSK: a tyrosine kinase of the Csk family that phosphorylates and inhibits Src family kinases. Specifically phosphorylates Y504 on LCK, which acts as a negative regulatory site. Csk and the phospho-tyrosine phosphatase CD45 reciprocally regulate phosphorylation of the inhibitory tyrosine of the Src family kinases Lck and Fyn during T-cell receptor mediated signaling during thymic development.
UniProt Protein Details:

Protein type:Protein kinase, tyrosine (non-receptor); EC 2.7.10.2; Protein kinase, TK; Kinase, protein; TK group; Csk family

Chromosomal Location of Human Ortholog: 15q24.1

Cellular Component: cytoplasm; cytosol; extrinsic to internal side of plasma membrane; plasma membrane

Molecular Function:identical protein binding; kinase activity; non-membrane spanning protein tyrosine kinase activity; protein binding; protein C-terminus binding; protein-tyrosine kinase activity; receptor binding

Biological Process: cell differentiation; cell migration; central nervous system development; epidermal growth factor receptor signaling pathway; innate immune response; negative regulation of Golgi to plasma membrane protein transport; protein amino acid phosphorylation; regulation of cell proliferation; regulation of cytokine production; T cell costimulation; T cell receptor signaling pathway; transmembrane receptor protein tyrosine kinase signaling pathway

UniProt Code:P41240
NCBI GenInfo Identifier:729887
NCBI Gene ID:1445
NCBI Accession:P41240.1
UniProt Secondary Accession:P41240,Q2M3N2, Q6FGZ6,
UniProt Related Accession:P41240
Molecular Weight:50,704 Da
NCBI Full Name:Tyrosine-protein kinase CSK
NCBI Synonym Full Names:c-src tyrosine kinase
NCBI Official Symbol:CSK  
NCBI Protein Information:tyrosine-protein kinase CSK
UniProt Protein Name:Tyrosine-protein kinase CSK
UniProt Synonym Protein Names:C-Src kinase; Protein-tyrosine kinase CYL
Protein Family:Chloroplast sensor kinase
UniProt Gene Name:CSK  
UniProt Entry Name:CSK_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-Csk (Phospho-Ser364) Antibody, Anti-Csk 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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