Phospho-Tyrosine Antibody (Mouse mAb) [N16H13] Datasheet

Biological Description

Specificity	Phospho-Tyrosine Antibody (Mouse mAb) [N16H13] detects a broad range of tyrosine-phosphorylated proteins and peptides.
Background	Phospho-tyrosine represents the O‑phosphorylated form of the amino acid tyrosine within proteins and functions as a central modular signal in eukaryotic regulatory networks, where its reversible formation and recognition by dedicated domains control many aspects of cell communication, growth, and differentiation. The modification resides on the phenolic hydroxyl of tyrosine in protein side chains and is generated and removed by a tripartite toolkit of protein tyrosine kinases and protein tyrosine phosphatases together with Src homology 2 and related phosphotyrosine-binding domains that read phospho-tyrosine motifs and assemble signaling complexes; this architecture forms a highly interconnected, combinatorial code that enables specific cellular responses to extracellular cues. Tyrosine phosphorylation in receptor and nonreceptor tyrosine kinases creates docking sites for SH2- and PTB-domain-containing adaptors and enzymes and thereby couples ligand-induced receptor activation to downstream cascades such as RAS–RAF–MEK–ERK, PI3K–AKT, JAK–STAT, and PLCγ–Ca²⁺ signaling, allowing phospho-tyrosine patterns on receptors and scaffolds to define pathway selection and signal amplitude. Large-scale analyses of human phospho-tyrosine networks show that sets of phospho-tyrosine motifs and SH2-domain specificities form interlinked protein clusters at activated receptors and within cytoplasmic signaling hubs, and conditional phospho-tyrosine–dependent interactions create pathway branching and feedback loops that coordinate receptor trafficking, cytoskeletal remodeling, transcriptional activation, and metabolic adaptation. The abundance and distribution of phospho-tyrosine sites are tightly controlled by the balance between kinases and classical protein tyrosine phosphatases, whose catalytic domains are highly selective for phospho-tyrosine and terminate or reshape signaling by dephosphorylating receptors, intermediates, and transcription factors. Alterations in this balance lead to persistent or defective signal propagation. Comparative genomic and evolutionary analyses indicate that the full phospho-tyrosine toolkit expanded markedly at the origin of multicellular animals, with coordinated increases in tyrosine kinases, phosphatases, and SH2-domain proteins that correlate with more complex cell–cell and cell–matrix communication, highlighting phospho-tyrosine as a key innovation for metazoan signaling complexity. Dysregulation of phospho-tyrosine signaling through mutations, overexpression, or aberrant activation of tyrosine kinases, and loss or misregulation of phosphatases, is a hallmark of many cancers, immune disorders, and metabolic diseases, in which altered phospho-tyrosine landscapes drive oncogenic signaling, resistance to apoptosis, aberrant immune activation, or impaired insulin and leptin responses.

Specificity

Phospho-Tyrosine Antibody (Mouse mAb) [N16H13] detects a broad range of tyrosine-phosphorylated proteins and peptides.

Background

Phospho-tyrosine represents the O‑phosphorylated form of the amino acid tyrosine within proteins and functions as a central modular signal in eukaryotic regulatory networks, where its reversible formation and recognition by dedicated domains control many aspects of cell communication, growth, and differentiation. The modification resides on the phenolic hydroxyl of tyrosine in protein side chains and is generated and removed by a tripartite toolkit of protein tyrosine kinases and protein tyrosine phosphatases together with Src homology 2 and related phosphotyrosine-binding domains that read phospho-tyrosine motifs and assemble signaling complexes; this architecture forms a highly interconnected, combinatorial code that enables specific cellular responses to extracellular cues. Tyrosine phosphorylation in receptor and nonreceptor tyrosine kinases creates docking sites for SH2- and PTB-domain-containing adaptors and enzymes and thereby couples ligand-induced receptor activation to downstream cascades such as RAS–RAF–MEK–ERK, PI3K–AKT, JAK–STAT, and PLCγ–Ca²⁺ signaling, allowing phospho-tyrosine patterns on receptors and scaffolds to define pathway selection and signal amplitude. Large-scale analyses of human phospho-tyrosine networks show that sets of phospho-tyrosine motifs and SH2-domain specificities form interlinked protein clusters at activated receptors and within cytoplasmic signaling hubs, and conditional phospho-tyrosine–dependent interactions create pathway branching and feedback loops that coordinate receptor trafficking, cytoskeletal remodeling, transcriptional activation, and metabolic adaptation. The abundance and distribution of phospho-tyrosine sites are tightly controlled by the balance between kinases and classical protein tyrosine phosphatases, whose catalytic domains are highly selective for phospho-tyrosine and terminate or reshape signaling by dephosphorylating receptors, intermediates, and transcription factors. Alterations in this balance lead to persistent or defective signal propagation. Comparative genomic and evolutionary analyses indicate that the full phospho-tyrosine toolkit expanded markedly at the origin of multicellular animals, with coordinated increases in tyrosine kinases, phosphatases, and SH2-domain proteins that correlate with more complex cell–cell and cell–matrix communication, highlighting phospho-tyrosine as a key innovation for metazoan signaling complexity. Dysregulation of phospho-tyrosine signaling through mutations, overexpression, or aberrant activation of tyrosine kinases, and loss or misregulation of phosphatases, is a hallmark of many cancers, immune disorders, and metabolic diseases, in which altered phospho-tyrosine landscapes drive oncogenic signaling, resistance to apoptosis, aberrant immune activation, or impaired insulin and leptin responses.

Usage Information

Application

WB, IP, IHC, IF, FCM

Dilution

WB	IP	IHC	IF	FCM
1:2000	1:100	1:2400 - 1:9600	1:1600 - 1:3200	1:1600 - 1:6400

Reactivity

All Species Expected

Source

Mouse Monoclonal Antibody

MW

Storage Buffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage
(from the date of receipt)

-20°C (avoid freeze-thaw cycles), 2 years

Experimental Methods
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. Reference Table for Selecting PVDF Membrane Pore Size Specifications ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Methods

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. Reference Table for Selecting PVDF Membrane Pore Size Specifications

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use primary antibody dilution buffer to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:2000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

References

https://pubmed.ncbi.nlm.nih.gov/22194470/
https://pubmed.ncbi.nlm.nih.gov/20813250/

Phospho-Tyrosine Antibody (Mouse mAb) [N16H13]

Biological Description

Usage Information

References

Application Data