Phospho-DDR1/DDR2 (Tyr796/740) Antibody (Mouse mAb) [M16G11]

Application: Reactivity: Human

Lane 1: HEK293 (hDDR2 transfected), Lane 2: HEK293 (hDDR2 transfected; Calyculin A, 100 nM, 10 min)

Usage Information

Dilution
WB1:5000

Application
WB

Reactivity
Human

Source
Mouse 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
97 kDa 120-130 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	MCF-7 cells; MCF-7 PAPP-A cells
Negative Control	MCF-7 PAPP-A/E483Q cells

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: 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 ( 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 1:5000), 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: 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 ( 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 1:5000), 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-DDR1/DDR2 (Tyr796/740) Antibody (Mouse mAb) [M16G11] detects endogenous levels of total DDR1/DDR2 protein only when it is phosphorylated at Tyr796/740.

Subcellular Location
Cell membrane, Membrane, Secreted

Uniprot ID
Q16832, Q08345

Clone
M16G11

Synonym(s)
6030432F18;AI323681;CAK;CD167;CD167a;DDR;DDR1;Drd1;EDDR1;HGK2;MCK10;Mpk6;NEP;NTRK4;PTK3;PTK3A;PTK3D;RTK6;TRKE

Background
Phospho‑DDR1/DDR2 (Tyr796/740) refers to the activated forms of the collagen receptors DDR1 and DDR2, two discoidin domain receptor tyrosine kinases that integrate extracellular matrix cues with intracellular signaling controlling adhesion, migration, and matrix remodeling. DDR1 carries a discoidin‑like collagen‑binding domain, a single transmembrane segment, a juxtamembrane region, and an intracellular kinase domain whose activation loop contains Tyr796; collagen engagement induces slow but sustained receptor autophosphorylation on multiple tyrosines, and antibodies specific for phospho‑Tyr796 detect DDR1 only in this ligand‐activated state, marking the productive kinase conformation that propagates downstream signaling. DDR2 shares a similar domain layout and is likewise activated by fibrillar collagens, but Tyr740 sits within its activation loop as a regulatory site that is phosphorylated not only during receptor autophosphorylation but also directly by Src family kinases, creating a key control point for DDR2 signaling output. Biochemical dissection of DDR2 shows that Src targets three activation‑loop residues (Tyr736, Tyr740, Tyr741); phosphorylation at Tyr740 stimulates intramolecular DDR2 autophosphorylation on additional cytoplasmic tyrosines, which then serve as docking sites for the adaptor Shc and assembly of DDR2–Shc signaling complexes, linking collagen engagement to Ras–MAPK and other Shc‑dependent pathways. Mutation of Tyr740 to phenylalanine relieves an inhibitory constraint within the activation loop, producing a receptor that displays collagen‑independent autophosphorylation and constitutive signaling, indicating that the unphosphorylated Tyr740 side chain restrains kinase activity and that its modification or substitution shifts DDR2 toward an active conformation. Across DDR1 and DDR2, phosphorylation at Tyr796 and Tyr740 therefore defines collagen‑ and Src‑responsive activation loop states that control receptor autophosphorylation, adaptor recruitment, and signal propagation into pathways regulating cell–matrix adhesion, motility, and tissue remodeling, and assays that specifically quantify phospho‑DDR1 (Tyr796) and phospho‑DDR2 (Tyr740) in lysates provide a direct biochemical readout of discoidin receptor engagement by collagen and of Src‑dependent tuning of DDR2 activity in physiological and pathological contexts.

Background

Phospho‑DDR1/DDR2 (Tyr796/740) refers to the activated forms of the collagen receptors DDR1 and DDR2, two discoidin domain receptor tyrosine kinases that integrate extracellular matrix cues with intracellular signaling controlling adhesion, migration, and matrix remodeling. DDR1 carries a discoidin‑like collagen‑binding domain, a single transmembrane segment, a juxtamembrane region, and an intracellular kinase domain whose activation loop contains Tyr796; collagen engagement induces slow but sustained receptor autophosphorylation on multiple tyrosines, and antibodies specific for phospho‑Tyr796 detect DDR1 only in this ligand‐activated state, marking the productive kinase conformation that propagates downstream signaling. DDR2 shares a similar domain layout and is likewise activated by fibrillar collagens, but Tyr740 sits within its activation loop as a regulatory site that is phosphorylated not only during receptor autophosphorylation but also directly by Src family kinases, creating a key control point for DDR2 signaling output. Biochemical dissection of DDR2 shows that Src targets three activation‑loop residues (Tyr736, Tyr740, Tyr741); phosphorylation at Tyr740 stimulates intramolecular DDR2 autophosphorylation on additional cytoplasmic tyrosines, which then serve as docking sites for the adaptor Shc and assembly of DDR2–Shc signaling complexes, linking collagen engagement to Ras–MAPK and other Shc‑dependent pathways. Mutation of Tyr740 to phenylalanine relieves an inhibitory constraint within the activation loop, producing a receptor that displays collagen‑independent autophosphorylation and constitutive signaling, indicating that the unphosphorylated Tyr740 side chain restrains kinase activity and that its modification or substitution shifts DDR2 toward an active conformation. Across DDR1 and DDR2, phosphorylation at Tyr796 and Tyr740 therefore defines collagen‑ and Src‑responsive activation loop states that control receptor autophosphorylation, adaptor recruitment, and signal propagation into pathways regulating cell–matrix adhesion, motility, and tissue remodeling, and assays that specifically quantify phospho‑DDR1 (Tyr796) and phospho‑DDR2 (Tyr740) in lysates provide a direct biochemical readout of discoidin receptor engagement by collagen and of Src‑dependent tuning of DDR2 activity in physiological and pathological contexts.

References
https://pubmed.ncbi.nlm.nih.gov/16186108/

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%