Choose Your Country or Region

Anti-Integrin β 3 Rabbit Antibody [P12C15]

Catalog No.: F3638

Application: Reactivity: Human, Mouse, Rat

Usage Information

Dilution
WB1:1000 IP1:20 FCM1:20

Application
WB, IP, FCM

Reactivity
Human, Mouse, Rat

Source
Rabbit

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
87 kDa

Positive Control	U-87MG cell; HEL cell; HUVEC cell; C2C12 cell
Negative Control	LnCaP cell; HeLa cell; PC-3 cell; Raw264.7 cell

Exprimental 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. 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, 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.

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.

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, 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
Integrin β 3 Rabbit mAb recognizes endogenous levels of total Integrin β 3 protein.

Subcellular Location
Cell junction, Cell membrane, Cell projection, Membrane, Postsynaptic cell membrane, Synapse

Uniprot ID
P05106

Clone
P12C15

Synonym(s)
CD61, GP3A, ITGB3, Integrin beta-3, Platelet membrane glycoprotein IIIa, GPIIIa

Background
Integrin β3 is a key subunit of the integrin family of transmembrane receptors that mediate cell adhesion to the extracellular matrix (ECM) and transduce signals bidirectionally across the plasma membrane. It commonly pairs with αV or αIIb subunits to form the αVβ3 and αIIbβ3 integrins, which play essential roles in diverse biological processes such as platelet aggregation (αIIbβ3), angiogenesis, cell migration, and survival (αVβ3). Integrin β3 features a large extracellular domain, including the βA (I-like) domain responsible for ligand binding, a hybrid domain, a plexin-semaphorin-integrin (PSI) domain, and multiple I-EGF domains, followed by a single transmembrane region and a short cytoplasmic tail that interacts with intracellular proteins. The transmembrane domain is critical for α/β heterodimerization and signal transmission. In its inactive state, β3 integrin adopts a bent conformation, but upon activation, it undergoes dramatic conformational changes, extension at the I-EGF1/2 "knee" and hybrid domain swing-out, enabling high-affinity ligand binding and separation of the legs. Activation is tightly regulated by inside-out signaling, where proteins like talin and kindlin bind to the cytoplasmic tail, triggering conformational shifts from low- to high-affinity states. Outside-in signaling follows ligand engagement, leading to cytoskeletal rearrangement and modulation of gene expression. β3 integrin also contributes to bone resorption by osteoclasts, immune cell trafficking, and tumor progression. Its activity is modulated by divalent cations (Ca²⁺, Mg²⁺, Mn²⁺), phosphorylation of cytoplasmic residues, and interactions with other membrane proteins. Genetic variants, such as the Leu33Pro polymorphism in the PSI domain, are associated with altered immune responses and increased risk of thrombosis. Dysregulation of β3 integrin is implicated in thrombosis, cancer metastasis, and inflammatory diseases.

Background

Integrin β3 is a key subunit of the integrin family of transmembrane receptors that mediate cell adhesion to the extracellular matrix (ECM) and transduce signals bidirectionally across the plasma membrane. It commonly pairs with αV or αIIb subunits to form the αVβ3 and αIIbβ3 integrins, which play essential roles in diverse biological processes such as platelet aggregation (αIIbβ3), angiogenesis, cell migration, and survival (αVβ3). Integrin β3 features a large extracellular domain, including the βA (I-like) domain responsible for ligand binding, a hybrid domain, a plexin-semaphorin-integrin (PSI) domain, and multiple I-EGF domains, followed by a single transmembrane region and a short cytoplasmic tail that interacts with intracellular proteins. The transmembrane domain is critical for α/β heterodimerization and signal transmission. In its inactive state, β3 integrin adopts a bent conformation, but upon activation, it undergoes dramatic conformational changes, extension at the I-EGF1/2 "knee" and hybrid domain swing-out, enabling high-affinity ligand binding and separation of the legs. Activation is tightly regulated by inside-out signaling, where proteins like talin and kindlin bind to the cytoplasmic tail, triggering conformational shifts from low- to high-affinity states. Outside-in signaling follows ligand engagement, leading to cytoskeletal rearrangement and modulation of gene expression. β3 integrin also contributes to bone resorption by osteoclasts, immune cell trafficking, and tumor progression. Its activity is modulated by divalent cations (Ca²⁺, Mg²⁺, Mn²⁺), phosphorylation of cytoplasmic residues, and interactions with other membrane proteins. Genetic variants, such as the Leu33Pro polymorphism in the PSI domain, are associated with altered immune responses and increased risk of thrombosis. Dysregulation of β3 integrin is implicated in thrombosis, cancer metastasis, and inflammatory diseases.

References
https://pubmed.ncbi.nlm.nih.gov/30018079/ https://pubmed.ncbi.nlm.nih.gov/19704023/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
If you have any other enquiries, please leave a message.

* Indicates a Required Field

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%