PAR2 Antibody [J17G12] | Primary Antibodies

Application: Reactivity: Human

Lane 1: HCT116, Lane 2: HepG2

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human

Source
Rabbit Monoclonal Antibody

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

Predicted MW
32 kDa

Positive Control	HCT 116 cells; Hep G2 cells
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 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 RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 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 RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. 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. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( 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 for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), 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.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

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 RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

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 RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. 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. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( 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 for 1 hour at room temperature;

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

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), 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.

Datasheet & SDS

Biological Description

Specificity
PAR2 Antibody [J17G12] detects endogenous levels of total PAR2 protein.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
P55085

Clone
J17G12

Synonym(s)
Proteinase-activated receptor 2; Coagulation factor II receptor-like 1; CD142; F2RL1

Background
PAR2 belongs to the protease-activated receptor family of G protein-coupled receptors alongside PAR1, PAR3, and PAR4, distinguished by proteolytic unmasking of cryptic N-terminal tethered ligands rather than diffusible agonists. PAR2 organizes seven transmembrane helices with an extracellular N-terminus featuring the SFLLRN sequence that, upon cleavage at Arg36 by trypsin-like serine proteases or mast cell tryptase, folds back to engage the second extracellular loop triggering G protein dissociation. Protease activation induces conformational shift where TM6 outward movement exposes DRY motif for Gαq/11 coupling that stimulates phospholipase Cβ hydrolysis of PIP2 into IP3 and DAG, mobilizing intracellular calcium for smooth muscle contraction alongside PKC-mediated Raf-MEK-ERK cascade activation driving cytokine transcription via Elk-1 and NF-κB nuclear translocation. β-arrestin-2 recruitment sustains MAPK signaling while promoting clathrin-mediated endocytosis that either recycles functional receptors or targets them for lysosomal degradation, with biased agonism from neutrophil elastase cleavage at distinct sites yielding G12/13-RhoA outputs favoring migration over secretion. Canonical trypsin cleavage at R36-S37 activates comprehensive Gq/11, Gi, and G12/13 pathways, while non-canonical sites produce signaling bias through differential β-arrestin engagement. PAR2 governs epithelial barrier restitution through Rac1-dependent lamellipodia protrusion and sensory neuron hyperexcitability via TRPV1 sensitization during inflammation, with highest expression in colon enterocytes, airway epithelium, and dorsal root ganglia reflecting roles in mucosal defense and pain transmission. Signal peptide shielding prevents premature intracellular activation during biosynthesis, ensuring surface competence. Constitutive PAR2 upregulation drives colitis-associated tumorigenesis and arthritic synovitis through IL-8 and MMP9 induction.

Background

PAR2 belongs to the protease-activated receptor family of G protein-coupled receptors alongside PAR1, PAR3, and PAR4, distinguished by proteolytic unmasking of cryptic N-terminal tethered ligands rather than diffusible agonists. PAR2 organizes seven transmembrane helices with an extracellular N-terminus featuring the SFLLRN sequence that, upon cleavage at Arg36 by trypsin-like serine proteases or mast cell tryptase, folds back to engage the second extracellular loop triggering G protein dissociation. Protease activation induces conformational shift where TM6 outward movement exposes DRY motif for Gαq/11 coupling that stimulates phospholipase Cβ hydrolysis of PIP2 into IP3 and DAG, mobilizing intracellular calcium for smooth muscle contraction alongside PKC-mediated Raf-MEK-ERK cascade activation driving cytokine transcription via Elk-1 and NF-κB nuclear translocation. β-arrestin-2 recruitment sustains MAPK signaling while promoting clathrin-mediated endocytosis that either recycles functional receptors or targets them for lysosomal degradation, with biased agonism from neutrophil elastase cleavage at distinct sites yielding G12/13-RhoA outputs favoring migration over secretion. Canonical trypsin cleavage at R36-S37 activates comprehensive Gq/11, Gi, and G12/13 pathways, while non-canonical sites produce signaling bias through differential β-arrestin engagement. PAR2 governs epithelial barrier restitution through Rac1-dependent lamellipodia protrusion and sensory neuron hyperexcitability via TRPV1 sensitization during inflammation, with highest expression in colon enterocytes, airway epithelium, and dorsal root ganglia reflecting roles in mucosal defense and pain transmission. Signal peptide shielding prevents premature intracellular activation during biosynthesis, ensuring surface competence. Constitutive PAR2 upregulation drives colitis-associated tumorigenesis and arthritic synovitis through IL-8 and MMP9 induction.

References
https://pubmed.ncbi.nlm.nih.gov/31733286/ https://pubmed.ncbi.nlm.nih.gov/41330898/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%