SYK (Phospho-Tyr323) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00033
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

SYK (Phospho-Tyr323) Cell-Based ELISA Kit

The SYK (Phospho-Tyr323) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor SYK phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SYK in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SYK phosphorylation.

How does our SYK (Phospho-Tyr323) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of SYK (Phospho-Tyr323) concentration is achieved by an indirect ELISA format. In essence, SYK (Phospho-Tyr323) is captured by SYK (Phospho-Tyr323)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2. An antibody against the nonphosphorylated counterpart of SYK (Phospho-Tyr323) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated SYK can be used to normalize the RFU value for phosphorylated SYK.

SYK (Phospho-Tyr323) Fluorometric Cell-Based ELISA Kit -Information

Product Name:SYK (Phospho-Tyr323) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00033
Description:The SYK (Phospho-Tyr323) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor SYK (Phospho-Tyr323) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SYK (Phospho-Tyr323) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on SYK phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 6850, UniProt ID: P43405, OMIM #: 600085, Unigene #: Hs.371720
Format:Two 96-Well Plates
NCBI Gene Symbol:SYK
Sub Type:Phospho
Target Name:Phospho-SYK (Tyr323)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components

Quantity

96-Well Black Cell CultureClear-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 Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
  • Micropipettes capable of measuring volumes from 1 µl to 1 ml
  • Deionized or sterile water (ddH2O)
  • 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)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.Dispense 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 3 minutes each time.
11.Add 50 µl of Primary Antibody Mixture P to corresponding wells for SYK (Phospho-Tyr323) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total SYK detection. Cover the plate 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 3 minutes each time.
13.Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

SYK (Phospho-Tyr323) - Protein Information

UniProt Protein Function:Syk: a cytoplasmic tyrosine kinase of the SYK family containing two SH2 domains. Plays a central role in the B cell receptor (BCR) response. An upstream activator of the PI3K, PLCgamma2, and Rac/cdc42 pathways in the BCR response. Required for the sequential events of Fc gamma IIa receptor-mediated phagocytosis. Expression highest in murine spleen, heart, mammary gland and thymus. Two splice variant isoforms have been described.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 9q22

Cellular Component: T cell receptor complex; extrinsic to internal side of plasma membrane; protein complex; cytoplasm; plasma membrane; nucleus; cytosol; B cell receptor complex

Molecular Function:integrin binding; protein serine/threonine kinase activity; protein binding; protein-tyrosine kinase activity; non-membrane spanning protein tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; protein kinase binding; ATP binding; protein kinase activity

Biological Process: viral reproduction; transcription factor import into nucleus; positive regulation of interleukin-3 biosynthetic process; positive regulation of granulocyte macrophage colony-stimulating factor biosynthetic process; positive regulation of calcium-mediated signaling; regulation of phagocytosis; protein amino acid phosphorylation; activation of JNK activity; leukocyte adhesion; B cell receptor signaling pathway; regulation of transcription factor activity; positive regulation of bone resorption; beta selection; positive regulation of gamma-delta T cell differentiation; angiogenesis; inflammatory response; integrin-mediated signaling pathway; platelet activation; neutrophil chemotaxis; adaptive immune response; regulation of superoxide release; positive regulation of cell adhesion mediated by integrin; positive regulation of mast cell degranulation; regulation of neutrophil degranulation; leukotriene biosynthetic process; cell proliferation; positive regulation of peptidyl-tyrosine phosphorylation; organ morphogenesis; neutrophil activation during immune response; serotonin secretion by platelet; leukocyte activation during immune response; defense response to bacterium; macrophage activation during immune response; positive regulation of B cell differentiation; blood vessel morphogenesis; innate immune response; positive regulation of alpha-beta T cell proliferation; lymph vessel development; blood coagulation; transmembrane receptor protein tyrosine kinase signaling pathway; positive regulation of alpha-beta T cell differentiation; positive regulation of cytokine secretion

NCBI Summary:This gene encodes a member of the family of non-receptor type Tyr protein kinases. This protein is widely expressed in hematopoietic cells and is involved in coupling activated immunoreceptors to downstream signaling events that mediate diverse cellular responses, including proliferation, differentiation, and phagocytosis. It is thought to be a modulator of epithelial cell growth and a potential tumour suppressor in human breast carcinomas. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Mar 2010]
UniProt Code:P43405
NCBI GenInfo Identifier:1174527
NCBI Gene ID:6850
NCBI Accession:P43405.1
UniProt Related Accession:P43405
Molecular Weight:635
NCBI Full Name:Tyrosine-protein kinase SYK
NCBI Synonym Full Names:spleen tyrosine kinase
NCBI Official Symbol:SYK  
NCBI Official Synonym Symbols:p72-Syk  
NCBI Protein Information:tyrosine-protein kinase SYK
UniProt Protein Name:Tyrosine-protein kinase SYK
UniProt Synonym Protein Names:Spleen tyrosine kinase; p72-Syk
Protein Family:Tyrosine-protein kinase
UniProt Gene Name:SYK  
UniProt Entry Name:KSYK_HUMAN