Toll-like Receptor 2 Antibody [M20D11]

Application: Reactivity: Mouse

Lane 1: RAW264.7, Lane 2: BMDC

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Mouse

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
85-100 kDa

Positive Control	Raw 264.7 cells; BMDM cells; BMDC 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. 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) 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 RIPA/NP-40 Lysis Buffer (containing Protease 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. 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, 120 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.

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) 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 RIPA/NP-40 Lysis Buffer (containing Protease 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.

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, 120 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
Toll-like Receptor 2 Antibody [M20D11] detects endogenous levels of total Toll-like Receptor 2 protein.

Subcellular Location
Cell membrane, Cytoplasmic vesicle, Membrane

Uniprot ID
Q9QUN7

Clone
M20D11

Synonym(s)
Toll-like receptor 2; CD282; Tlr2

Background
Toll-like receptor 2, or TLR2, is a type I transmembrane glycoprotein in the TLR family of pattern recognition receptors that primarily acts as a sentinel for microbial components on the cell surfaces of macrophages, dendritic cells, and neutrophils. It recognizes a wide range of pathogen-associated molecular patterns through obligatory heterodimerization with TLR1, which detects triacylated lipopeptides, or TLR6, which detects diacylated lipopeptides, together forming m-shaped ligand-binding platforms. The extracellular domain of TLR2 has a horseshoe shape composed of 10 leucine-rich repeats, LRRs 11-20, forming continuous beta-strands and a convex scaffold stabilized by disulfide bonds, followed by a transmembrane helix and a cytoplasmic Toll/IL-1 receptor, TIR, domain of about 150 amino acids. The TIR domain features a BB-loop, with Pro681 and His686 critical for MyD88 adaptor docking, and oligomerizes upon ligand-induced dimerization, bringing TIRs within about 5 nanometers of each other. TLR2 is ligand binding, with Pam3CSK4 and Pam2CSK4 occupying hydrophobic patches on the LRRs, triggering a receptor conformational switch that recruits TIRAP and MyD88 adaptors through TIR-TIR interfaces. This activates IRAK4, IRAK1, and TRAF6, leading to TAK1 activation and the IKKβ/NF-κB cascade, which induces pro-inflammatory cytokines such as TNFα, IL-6, and IL-12, as well as MAPK signaling via p38, JNK, and ERK1/2, and antimicrobial responses, while also facilitating phagocytosis and endosomal maturation. TLR2-positive monocytes show enhanced endothelial adhesion and migration through ICAM-1 upregulation independently of inflammation. TLR2 bridges innate and adaptive immunity by promoting dendritic cell maturation and CD4+ T cell priming, enforces immune tolerance through Treg expansion and Pam3CSK4-driven resistance to IL-2-mediated suppression, and maintains homeostasis by responding to endogenous ligands such as HMGB1 in sterile inflammation. Hyperactive TLR2 contributes to septic shock and atherosclerosis, where stimuli like oxLDL or Pam3CSK4 promote foam cell formation, while TLR2 deficiency impairs antifungal immunity against Aspergillus and Candida.

Background

Toll-like receptor 2, or TLR2, is a type I transmembrane glycoprotein in the TLR family of pattern recognition receptors that primarily acts as a sentinel for microbial components on the cell surfaces of macrophages, dendritic cells, and neutrophils. It recognizes a wide range of pathogen-associated molecular patterns through obligatory heterodimerization with TLR1, which detects triacylated lipopeptides, or TLR6, which detects diacylated lipopeptides, together forming m-shaped ligand-binding platforms. The extracellular domain of TLR2 has a horseshoe shape composed of 10 leucine-rich repeats, LRRs 11-20, forming continuous beta-strands and a convex scaffold stabilized by disulfide bonds, followed by a transmembrane helix and a cytoplasmic Toll/IL-1 receptor, TIR, domain of about 150 amino acids. The TIR domain features a BB-loop, with Pro681 and His686 critical for MyD88 adaptor docking, and oligomerizes upon ligand-induced dimerization, bringing TIRs within about 5 nanometers of each other. TLR2 is ligand binding, with Pam3CSK4 and Pam2CSK4 occupying hydrophobic patches on the LRRs, triggering a receptor conformational switch that recruits TIRAP and MyD88 adaptors through TIR-TIR interfaces. This activates IRAK4, IRAK1, and TRAF6, leading to TAK1 activation and the IKKβ/NF-κB cascade, which induces pro-inflammatory cytokines such as TNFα, IL-6, and IL-12, as well as MAPK signaling via p38, JNK, and ERK1/2, and antimicrobial responses, while also facilitating phagocytosis and endosomal maturation. TLR2-positive monocytes show enhanced endothelial adhesion and migration through ICAM-1 upregulation independently of inflammation. TLR2 bridges innate and adaptive immunity by promoting dendritic cell maturation and CD4+ T cell priming, enforces immune tolerance through Treg expansion and Pam3CSK4-driven resistance to IL-2-mediated suppression, and maintains homeostasis by responding to endogenous ligands such as HMGB1 in sterile inflammation. Hyperactive TLR2 contributes to septic shock and atherosclerosis, where stimuli like oxLDL or Pam3CSK4 promote foam cell formation, while TLR2 deficiency impairs antifungal immunity against Aspergillus and Candida.

References
https://pubmed.ncbi.nlm.nih.gov/37569837/ https://pubmed.ncbi.nlm.nih.gov/22566960/

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

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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%