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SHP-1 (Phospho-Tyr564) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00087
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

SHP-1 (Phospho-Tyr564) Cell-Based ELISA Kit

The SHP-1 (Phospho-Tyr564) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor SHP-1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SHP-1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SHP-1 phosphorylation.

How does our SHP-1 (Phospho-Tyr564) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of SHP-1 (Phospho-Tyr564) concentration is achieved by an indirect ELISA format. In essence, SHP-1 (Phospho-Tyr564) is captured by SHP-1 (Phospho-Tyr564)-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 SHP-1 (Phospho-Tyr564) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated SHP-1 can be used to normalize the RFU value for phosphorylated SHP-1.

SHP-1 (Phospho-Tyr564) Fluorometric Cell-Based ELISA Kit -Information

Product Name:SHP-1 (Phospho-Tyr564) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00087
Description:The SHP-1 (Phospho-Tyr564) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor SHP-1 (Phospho-Tyr564) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SHP-1 (Phospho-Tyr564) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on PTPN6 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 5777, UniProt ID: P29350, OMIM #: 176883, Unigene #: Hs.63489
Format:Two 96-Well Plates
NCBI Gene Symbol:PTPN6
Sub Type:Phospho
Target Name:Phospho-SHP-1 (Tyr564)

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 SHP-1 (Phospho-Tyr564) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total SHP-1 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.

SHP-1 (Phospho-Tyr564) - Protein Information

UniProt Protein Function:Modulates signaling by tyrosine phosphorylated cell surface receptors such as KIT and the EGF receptor/EGFR. The SH2 regions may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. Together with MTUS1, induces UBE2V2 expression upon angiotensin II stimulation. Plays a key role in hematopoiesis.
NCBI Summary:The protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. N-terminal part of this PTP contains two tandem Src homolog (SH2) domains, which act as protein phospho-tyrosine binding domains, and mediate the interaction of this PTP with its substrates. This PTP is expressed primarily in hematopoietic cells, and functions as an important regulator of multiple signaling pathways in hematopoietic cells. This PTP has been shown to interact with, and dephosphorylate a wide spectrum of phospho-proteins involved in hematopoietic cell signaling. Multiple alternatively spliced variants of this gene, which encode distinct isoforms, have been reported. [provided by RefSeq, Jul 2008]
UniProt Code:P29350
NCBI GenInfo Identifier:131469
NCBI Gene ID:5777
NCBI Accession:P29350.1
UniProt Secondary Accession:P29350,Q969V8, Q9UK67, A8K306, G3V0F8,
UniProt Related Accession:P29350
Molecular Weight:64 kDa
NCBI Full Name:Tyrosine-protein phosphatase non-receptor type 6
NCBI Synonym Full Names:protein tyrosine phosphatase, non-receptor type 6
NCBI Official Symbol:PTPN6  
NCBI Official Synonym Symbols:HCP; HCPH; SHP1; SHP-1; HPTP1C; PTP-1C; SHP-1L; SH-PTP1  
NCBI Protein Information:tyrosine-protein phosphatase non-receptor type 6
UniProt Protein Name:Tyrosine-protein phosphatase non-receptor type 6
UniProt Synonym Protein Names:Hematopoietic cell protein-tyrosine phosphatase; Protein-tyrosine phosphatase 1C; PTP-1C; Protein-tyrosine phosphatase SHP-1; SH-PTP1
Protein Family:Tyrosine-protein phosphatase non-receptor
UniProt Gene Name:PTPN6