Phospho-ALK (Tyr1278) Antibody (Rabbit mAb) [N16F10]

Application: Reactivity: Human

Lane 1: KARPAS-299, Lane 2: KARPAS-299 (phosphatase treated membrane)

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:100

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 (NPM-ALK); 220 kDa (ALK)
^*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	SUP-M2 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, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 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) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 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) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min. 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 ( 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 primary antibody dilution buffer 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

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) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

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) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.

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 ( 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 primary antibody dilution buffer 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 (Tyr1278) Antibody (Rabbit mAb) [N16F10] detects endogenous levels of total ALK protein only when it is phosphorylated at Tyr1278.

Subcellular Location
Cell membrane, Membrane

Uniprot ID
Q9UM73

Clone
N16F10

Synonym(s)
ALK; ALK receptor tyrosine kinase; ALK tyrosine kinase receptor; CD246; CD246 antigen; mutant anaplastic lymphoma kinase; NBLST3

Background
Phospho ALK (Tyr1278) designates the activated form of the receptor tyrosine kinase ALK within its activation loop, a state that underlies oncogenic signaling from full length ALK and ALK fusion proteins in diverse tumors. ALK belongs to the insulin receptor superfamily and contains an extracellular ligand binding region, a single transmembrane helix, and an intracellular kinase domain whose activation loop harbors Tyr1278 together with neighboring tyrosines that undergo autophosphorylation when ligand binding or oncogenic dimerization brings ALK molecules into proximity, converting the kinase from a low activity to a high activity state and enabling efficient phosphorylation of substrates. Substitution of Tyr1278 by polar or charged residues generates ALK variants with constitutive kinase activation, increased autophosphorylation, and ligand independent transforming activity, indicating that phosphorylation or mutation at this position stabilizes the active conformation of the activation loop and removes an autoinhibitory constraint on the catalytic cleft. Activated, Tyr1278 phosphorylated ALK triggers multiple downstream cascades, including PI3K–AKT, RAS–ERK, JAK–STAT, and PLCγ pathways, through phosphorylation of adaptor and docking proteins such as IRS 1, Shc, PLCγ, and other substrates identified by phosphoproteomic profiling, and these pathways together drive proliferation, survival, migration, and transformation in ALK positive tumor cells. Oncogenic ALK activation arises through chromosomal translocations that fuse the ALK kinase domain to oligomerization competent partners such as NPM1 in anaplastic large cell lymphoma and EML4 in non small cell lung cancer, through activating point mutations and amplification in neuroblastoma, and through aberrant overexpression in several additional malignancies; in each setting, constitutive phosphorylation of the activation loop, including Tyr1278, is a biochemical hallmark of the oncogenic kinase state. Phospho specific antibodies that recognize ALK only when phosphorylated at Tyr1278, a site equivalent to Tyr338 in NPM ALK, detect this modification in ALK driven carcinoma cell lines and primary tumors and provide a direct readout of ALK catalytic engagement, complementing genetic assays that identify ALK rearrangements or mutations.

Background

Phospho ALK (Tyr1278) designates the activated form of the receptor tyrosine kinase ALK within its activation loop, a state that underlies oncogenic signaling from full length ALK and ALK fusion proteins in diverse tumors. ALK belongs to the insulin receptor superfamily and contains an extracellular ligand binding region, a single transmembrane helix, and an intracellular kinase domain whose activation loop harbors Tyr1278 together with neighboring tyrosines that undergo autophosphorylation when ligand binding or oncogenic dimerization brings ALK molecules into proximity, converting the kinase from a low activity to a high activity state and enabling efficient phosphorylation of substrates. Substitution of Tyr1278 by polar or charged residues generates ALK variants with constitutive kinase activation, increased autophosphorylation, and ligand independent transforming activity, indicating that phosphorylation or mutation at this position stabilizes the active conformation of the activation loop and removes an autoinhibitory constraint on the catalytic cleft. Activated, Tyr1278 phosphorylated ALK triggers multiple downstream cascades, including PI3K–AKT, RAS–ERK, JAK–STAT, and PLCγ pathways, through phosphorylation of adaptor and docking proteins such as IRS 1, Shc, PLCγ, and other substrates identified by phosphoproteomic profiling, and these pathways together drive proliferation, survival, migration, and transformation in ALK positive tumor cells. Oncogenic ALK activation arises through chromosomal translocations that fuse the ALK kinase domain to oligomerization competent partners such as NPM1 in anaplastic large cell lymphoma and EML4 in non small cell lung cancer, through activating point mutations and amplification in neuroblastoma, and through aberrant overexpression in several additional malignancies; in each setting, constitutive phosphorylation of the activation loop, including Tyr1278, is a biochemical hallmark of the oncogenic kinase state. Phospho specific antibodies that recognize ALK only when phosphorylated at Tyr1278, a site equivalent to Tyr338 in NPM ALK, detect this modification in ALK driven carcinoma cell lines and primary tumors and provide a direct readout of ALK catalytic engagement, complementing genetic assays that identify ALK rearrangements or mutations.

References
https://pubmed.ncbi.nlm.nih.gov/19459784/ https://pubmed.ncbi.nlm.nih.gov/29084134/

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%