Phospho-ALK (Tyr1604) Antibody [G21H21]

Application: Reactivity: Human

Lane 1: KARPAS-299, Lane 2: KARPAS-299 (pervanadate, 1 mM, 30 min)

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:30

Application
WB, IP

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 Observed MW
176 kDa 80 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	KARPAS-299 cells (Pervanadate, 1 mM, 30 min)
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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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, Phosphatase 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, Phosphatase 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, Phosphatase 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 ( recommending 5% BSA 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
Phospho-ALK (Tyr1604) Antibody [G21H21] detects endogenous levels of total ALK protein only when it is phosphorylated at Tyr1604.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
Q9UM73

Clone
G21H21

Synonym(s)
CD246; ALK tyrosine kinase receptor; Anaplastic lymphoma kinase; ALK

Background
Phospho-ALK (Tyr1604) belongs to the insulin receptor tyrosine kinase family and serves as a receptor for ligands like ALKAL1 and ALKAL2, which drive its activation in neural development and energy homeostasis. The protein spans 1620 amino acids with an extracellular ligand-binding region featuring MAM and glycine-rich domains, a single transmembrane helix, and an intracellular kinase domain where Tyr1604 resides in the activation loop. Upon ligand binding, ALK dimerizes, leading to autophosphorylation at multiple tyrosines, including Tyr1604, which fully activates the kinase and enables docking of effectors like PLCγ. Phosphorylation at Tyr1604 proves essential for PLCγ recruitment, triggering PLCγ activation that generates IP3 and DAG to mobilize calcium and activate PKC, fueling cell proliferation and survival signals. This site corresponds to Tyr664 in the oncogenic NPM-ALK fusion, where mutation abolishes PLCγ binding, oncogenic potency, and mitogenic activity in anaplastic large cell lymphoma. ALK signaling through Tyr1604 phosphorylation propagates downstream to MAPK/ERK, PI3K/AKT, and NF-κB pathways, promoting neuronal differentiation, anti-apoptotic responses, and autocrine growth in MDK/PTN-stimulated cells. In non-small cell lung cancer, EML4-ALK fusions cause ligand-independent Tyr1604 phosphorylation, hyperactivating these pathways to drive tumorigenesis, invasion, and resistance to apoptosis. Phosphorylated ALK at Tyr1604 in hypothalamic neurons curbs lipolysis and sympathetic outflow, enhancing resistance to diet-induced obesity. Dysregulated Tyr1604 phosphorylation underlies aggressive ALK-driven malignancies like neuroblastoma and inflammatory myofibroblastic tumors, where it sustains PLCγ-mediated pathogenesis.

Background

Phospho-ALK (Tyr1604) belongs to the insulin receptor tyrosine kinase family and serves as a receptor for ligands like ALKAL1 and ALKAL2, which drive its activation in neural development and energy homeostasis. The protein spans 1620 amino acids with an extracellular ligand-binding region featuring MAM and glycine-rich domains, a single transmembrane helix, and an intracellular kinase domain where Tyr1604 resides in the activation loop. Upon ligand binding, ALK dimerizes, leading to autophosphorylation at multiple tyrosines, including Tyr1604, which fully activates the kinase and enables docking of effectors like PLCγ. Phosphorylation at Tyr1604 proves essential for PLCγ recruitment, triggering PLCγ activation that generates IP3 and DAG to mobilize calcium and activate PKC, fueling cell proliferation and survival signals. This site corresponds to Tyr664 in the oncogenic NPM-ALK fusion, where mutation abolishes PLCγ binding, oncogenic potency, and mitogenic activity in anaplastic large cell lymphoma. ALK signaling through Tyr1604 phosphorylation propagates downstream to MAPK/ERK, PI3K/AKT, and NF-κB pathways, promoting neuronal differentiation, anti-apoptotic responses, and autocrine growth in MDK/PTN-stimulated cells. In non-small cell lung cancer, EML4-ALK fusions cause ligand-independent Tyr1604 phosphorylation, hyperactivating these pathways to drive tumorigenesis, invasion, and resistance to apoptosis. Phosphorylated ALK at Tyr1604 in hypothalamic neurons curbs lipolysis and sympathetic outflow, enhancing resistance to diet-induced obesity. Dysregulated Tyr1604 phosphorylation underlies aggressive ALK-driven malignancies like neuroblastoma and inflammatory myofibroblastic tumors, where it sustains PLCγ-mediated pathogenesis.

References
https://pubmed.ncbi.nlm.nih.gov/23889739/

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%