PTK7/CCK4 Antibody [B13K12] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: A204, Lane 2: C2C12, Lane 3: PC12

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:200

Application
WB, IP

Reactivity
Human, Mouse, Rat

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
160 kDa

Positive Control	A204 cells; SNB19 cells; mIMCD-3 cells; C2C12 cells; PC-12 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
PTK7/CCK4 Antibody [B13K12] detects endogenous levels of total PTK7/CCK4 protein.

Subcellular Location
Cell junction, Membrane

Uniprot ID
Q13308

Clone
B13K12

Synonym(s)
Inactive tyrosine-protein kinase 7; Colon carcinoma kinase 4 (CCK-4); Protein-tyrosine kinase 7; Pseudo tyrosine kinase receptor 7; Tyrosine-protein kinase-like 7; PTK7; CCK4

Background
PTK7, also known as CCK4, belongs to the receptor tyrosine kinase family despite lacking catalytic kinase activity due to missing key residues in its kinase-like domain. The protein spans about 1070 amino acids, featuring an extracellular region with seven immunoglobulin-like (Ig)-like domains (loops 1-7), a single transmembrane helix, and a 345-residue intracellular portion with a sterile alpha motif (SAM) and catalytically inactive tyrosine kinase homology domain. Ig domains 1-7 mediate ligand-independent homodimerization and heterotypic interactions, while the kinase domain, absent the DFG motif for Mg2+ chelation, scaffolds adaptor binding. PTK7 orchestrates planar cell polarity (PCP) by recruiting Dishevelled (Dsh) via its PDZ-binding motif to Frizzled receptors, activating non-canonical Wnt/PCP signaling with RACK1 and Dsh for convergent extension, neural tube closure, and neural crest migration. In canonical Wnt, PTK7 competes with Frizzled for binding, suppressing beta-catenin stabilization and transcription. Proteolytic cleavage by MMP14 (between Pro621-Leu622) and ADAM17 sheds the extracellular domain, releasing a soluble fragment that boosts proliferation, migration, and invasion while generating a membrane stub for enhanced signaling. PTK7 governs cell adhesion, polarity, motility, actin reorganization, and apoptosis during embryogenesis, epithelial organization, axon guidance, and angiogenesis. It co-receptors with Plexin-A1 in semaphorin signaling for cytoskeletal dynamics and VEGFR1 in vascular sprouting. In cancer, upregulated PTK7 drives metastasis in colon, gastric, lung, and ovarian tumors via PCP activation and EGFR stabilization; the V354M germline variant elevates levels, inhibits p53/p21/CREB, and boosts AKT to promote proliferation. PTK7 marks poor prognosis in acute myeloid leukemia and serves as an antibody-drug conjugate target. Membrane trafficking and Cdx-regulated expression fine-tune its roles in development and oncogenesis.

Background

PTK7, also known as CCK4, belongs to the receptor tyrosine kinase family despite lacking catalytic kinase activity due to missing key residues in its kinase-like domain. The protein spans about 1070 amino acids, featuring an extracellular region with seven immunoglobulin-like (Ig)-like domains (loops 1-7), a single transmembrane helix, and a 345-residue intracellular portion with a sterile alpha motif (SAM) and catalytically inactive tyrosine kinase homology domain. Ig domains 1-7 mediate ligand-independent homodimerization and heterotypic interactions, while the kinase domain, absent the DFG motif for Mg2+ chelation, scaffolds adaptor binding. PTK7 orchestrates planar cell polarity (PCP) by recruiting Dishevelled (Dsh) via its PDZ-binding motif to Frizzled receptors, activating non-canonical Wnt/PCP signaling with RACK1 and Dsh for convergent extension, neural tube closure, and neural crest migration. In canonical Wnt, PTK7 competes with Frizzled for binding, suppressing beta-catenin stabilization and transcription. Proteolytic cleavage by MMP14 (between Pro621-Leu622) and ADAM17 sheds the extracellular domain, releasing a soluble fragment that boosts proliferation, migration, and invasion while generating a membrane stub for enhanced signaling. PTK7 governs cell adhesion, polarity, motility, actin reorganization, and apoptosis during embryogenesis, epithelial organization, axon guidance, and angiogenesis. It co-receptors with Plexin-A1 in semaphorin signaling for cytoskeletal dynamics and VEGFR1 in vascular sprouting. In cancer, upregulated PTK7 drives metastasis in colon, gastric, lung, and ovarian tumors via PCP activation and EGFR stabilization; the V354M germline variant elevates levels, inhibits p53/p21/CREB, and boosts AKT to promote proliferation. PTK7 marks poor prognosis in acute myeloid leukemia and serves as an antibody-drug conjugate target. Membrane trafficking and Cdx-regulated expression fine-tune its roles in development and oncogenesis.

References
https://pubmed.ncbi.nlm.nih.gov/22230326/ https://pubmed.ncbi.nlm.nih.gov/28424771/

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%