EphA3/A4/A5 Antibody [A18J10] | Primary Antibodies

Application: Reactivity: Human, Mouse

Lane 1: MOLT-4, Lane 2: NIH/3T3

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000 IP1:100

Application
WB, IP

Reactivity
Human, 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
135 kDa

Positive Control	SNB19 cells; MOLT-4 cells; mIMCD-3 cells; NIH/3T3 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. 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: 5%. 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.

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: 5%. 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
EphA3/A4/A5 Antibody [A18J10] detects endogenous levels of total EphA3, EphA4, and EphA5 proteins.

Subcellular Location
Cell membrane, Cell projection, Secreted, Cell junction, Endosome, Postsynaptic cell membrane, Synapse

Uniprot ID
P54756; P29320; P54764

Clone
A18J10

Synonym(s)
Ephrin type-A receptor 3/4/5; EPHA3; EPHA4; EPHA5

Background
EphA3/A4/A5 receptors belong to the Eph family of receptor tyrosine kinases, specifically the EphA subclass that preferentially engages GPI-anchored ephrin-A ligands to orchestrate cell-cell repulsion and topographic mapping during development. These receptors share an extracellular ligand-binding domain, cysteine-rich region, and intracellular kinase domain with SAM and PDZ-binding motifs that recruit adaptors like Nck and Crk for signal propagation. Ligand binding induces receptor clustering and autophosphorylation, activating PI3K-AKT and MAPK/ERK cascades alongside Rho GTPase modulation through ephexin and Vav exchange factors that drive actin cytoskeleton contraction and lamellipodial retraction essential for contact repulsion. EphA3 preferentially pairs with ephrin-A5 through a tilted high-affinity interface where the ephrin G-H loop inserts into the receptor pocket, stabilizing a conformation that enhances kinase activation compared to EphA2 while EphA4 and A5 exhibit overlapping specificities that fine-tune retinotectal projections and callosal axon guidance via graded signaling gradients. Forward signaling suppresses AKT to promote apoptosis and cell cycle arrest in tumor contexts, while somatic mutations in kinase or ligand-binding domains disrupt this tumor-suppressive function, permitting unchecked proliferation and metastasis in lung, colorectal, and breast cancers. EphA3 governs cardiac mesenchyme migration, and EphA4 coordinates hindbrain segmentation alongside vascular patterning through repulsive cues that prevent aberrant tissue fusion. Dysregulation through inactivating mutations correlates with lung cancer progression and angiogenesis in gastric carcinoma, while EphA5 maintains topographic retinal projections.

Background

EphA3/A4/A5 receptors belong to the Eph family of receptor tyrosine kinases, specifically the EphA subclass that preferentially engages GPI-anchored ephrin-A ligands to orchestrate cell-cell repulsion and topographic mapping during development. These receptors share an extracellular ligand-binding domain, cysteine-rich region, and intracellular kinase domain with SAM and PDZ-binding motifs that recruit adaptors like Nck and Crk for signal propagation. Ligand binding induces receptor clustering and autophosphorylation, activating PI3K-AKT and MAPK/ERK cascades alongside Rho GTPase modulation through ephexin and Vav exchange factors that drive actin cytoskeleton contraction and lamellipodial retraction essential for contact repulsion. EphA3 preferentially pairs with ephrin-A5 through a tilted high-affinity interface where the ephrin G-H loop inserts into the receptor pocket, stabilizing a conformation that enhances kinase activation compared to EphA2 while EphA4 and A5 exhibit overlapping specificities that fine-tune retinotectal projections and callosal axon guidance via graded signaling gradients. Forward signaling suppresses AKT to promote apoptosis and cell cycle arrest in tumor contexts, while somatic mutations in kinase or ligand-binding domains disrupt this tumor-suppressive function, permitting unchecked proliferation and metastasis in lung, colorectal, and breast cancers. EphA3 governs cardiac mesenchyme migration, and EphA4 coordinates hindbrain segmentation alongside vascular patterning through repulsive cues that prevent aberrant tissue fusion. Dysregulation through inactivating mutations correlates with lung cancer progression and angiogenesis in gastric carcinoma, while EphA5 maintains topographic retinal projections.

References
https://pubmed.ncbi.nlm.nih.gov/25993310/ https://pubmed.ncbi.nlm.nih.gov/25993310/

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%