Phospho-Src (Tyr419) Antibody [D21H6]

Application: Reactivity: Mouse, Rat, Human

Lane 1: A431, Lane 2: A431 (Pervanadate, 50 mM, 5 min), Lane 3: NIH/3T3, Lane 4: NIH/3T3 (Pervanadate, 50 mM, 5 min)

Usage Information

Dilution
WB1:5000 FCM1:100

Application
WB, FCM

Reactivity
Mouse, Rat, 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
60 kDa 60 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	A431 cells (pervanadate, 50 mM, 5 min); C6 (pervanadate, 50 mM, 5 min); NIH/3T3 (pervanadate, 50 mM, 5 min)
Negative Control	A431 cells; C6 cells; NIH/3T3 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. 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. 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 5% skim milk powder 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.

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.

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 5% skim milk powder 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-Src (Tyr419) Antibody [D21H6] detects endogenous levels of total Src protein only when it is phosphorylated at Tyr419.

Subcellular Location
Cell junction, Cell membrane, Cytoplasm, Cytoskeleton, Membrane, Mitochondrion, Mitochondrion inner membrane, Nucleus

Uniprot ID
P12931

Clone
D21H6

Synonym(s)
SRC1; SRC; Proto-oncogene tyrosine-protein kinase Src; Proto-oncogene c-Src; pp60c-src; p60-Src

Background
Phospho-Src (Tyr419), the activation loop phosphorylation marking full catalytic competence of the Src family kinase prototype, unlocks bidirectional tyrosine kinase signaling from virtually all integrin, growth factor, and GPCR stimuli across adherent cells and platelets. Dephosphorylation of inhibitory Tyr530 by CD45/PTP1B relieves SH2 domain clamping, permitting trans-autophosphorylation at Tyr419 that rigidly positions the activation loop for substrate access while relieving steric occlusion of the ATP-binding cleft; this catalyzes hierarchical phosphorylation cascades where Src primes receptor tyrosine kinases through Y1009/1101 autophosphorylation on EGFR while amplifying FAK at Y397/576/577/861 to scaffold paxillin/PI3K recruitment. Active p-Tyr419-Src drives Ras-GRF1/ERK for proliferation, STAT3/5a for survival, and cortactin/pyk2 for invadopodia maturation via sequential Arp2/3 activation; feedback engages Csk/Shp1 through C-terminal docking to restore Tyr530 phosphorylation, while PP2A/PP1 dephosphorylate the activation loop for signal termination. In platelets, shear-induced clustering at podosome-like adhesions generates local p-Tyr419 gradients that license αIIbβ3 outside-in signaling for clot retraction, while fibroblasts exploit Src for durotaxis through myosin-IIA/Y1183 feedback with tension sensors.

Background

Phospho-Src (Tyr419), the activation loop phosphorylation marking full catalytic competence of the Src family kinase prototype, unlocks bidirectional tyrosine kinase signaling from virtually all integrin, growth factor, and GPCR stimuli across adherent cells and platelets. Dephosphorylation of inhibitory Tyr530 by CD45/PTP1B relieves SH2 domain clamping, permitting trans-autophosphorylation at Tyr419 that rigidly positions the activation loop for substrate access while relieving steric occlusion of the ATP-binding cleft; this catalyzes hierarchical phosphorylation cascades where Src primes receptor tyrosine kinases through Y1009/1101 autophosphorylation on EGFR while amplifying FAK at Y397/576/577/861 to scaffold paxillin/PI3K recruitment. Active p-Tyr419-Src drives Ras-GRF1/ERK for proliferation, STAT3/5a for survival, and cortactin/pyk2 for invadopodia maturation via sequential Arp2/3 activation; feedback engages Csk/Shp1 through C-terminal docking to restore Tyr530 phosphorylation, while PP2A/PP1 dephosphorylate the activation loop for signal termination. In platelets, shear-induced clustering at podosome-like adhesions generates local p-Tyr419 gradients that license αIIbβ3 outside-in signaling for clot retraction, while fibroblasts exploit Src for durotaxis through myosin-IIA/Y1183 feedback with tension sensors.

References
https://pubmed.ncbi.nlm.nih.gov/9988270/ https://pubmed.ncbi.nlm.nih.gov/23923048/

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%