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Phospho-VASP (Ser 157) Rabbit mAb

Catalog No.: F1397

Lane 1: A-431
Lane 2: A-431 (Forskolin, 10 μM, 15 min)

Experiment Essentials

Subcellular Location: Cell junction, Cell membrane, Cell projection, Cytoplasm, Cytoskeleton, Membrane.
WB
Recommending using RIPA/NP-40 Lysis Buffer to prepare lysates.

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Source
Rabbit

Reactivity
Human, Mouse, Rat

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
50 kDa

Positive Control	A-431 (treat with Forskolin, 10 μM, 15 min); 3T3 (treat with Forskolin, 10 μM, 15 min)
Negative Control	A-431; 3T3

Exprimental 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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. 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 1:1000), 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 1172. 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.

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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add 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.Add 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. 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 1:1000), 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

1172. 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-VASP (Ser157) Rabbit mAb recognizes endogenous levels of VASP protein only when phosphorylated at Ser157.

Synonym(s)
Phospho-VASP (Ser157),VASP (phospho S157)

Uniprot ID
P50552

Clone
G6G22

Background
Vasodilator-stimulated phosphoprotein (VASP) is an actin- and profilin-binding protein highly expressed in platelets, where it plays a key role in suppressing secretory and adhesive events. VASP is a major substrate for protein kinases regulated by cAMP and cGMP, such as protein kinase A (PKA) and protein kinase G (PKG). Additionally, it is directly phosphorylated at Ser157 by protein kinase C (PKC). Originally identified in platelets, VASP is phosphorylated in response to vasodilators like prostaglandin I2 (PGI2) and nitric oxide (NO), which increase intracellular cAMP and cGMP levels. Ena/VASP family proteins, including VASP, share a conserved structure featuring an N-terminal EVH1 (Ena/VASP-homology-1) domain, a central proline-rich region, and a C-terminal EVH2 domain. In vivo studies indicate that VASP negatively regulates platelet function. Ena/VASP proteins are essential for cell motility, migration, and adhesion. VASP contributes to profilin recruitment, actin nucleation, bundling, and filament formation, and it is thought to have anti-branching and anti-capping roles in actin dynamics. VASP localizes to key cytoskeletal and adhesive structures, including the leading edge of lamellipodia, actin stress fibers, filopodial tips, and focal adhesions, such as those involving the integrin αIIbβ3 in platelets. Phosphorylation of VASP occurs at three specific sites—Ser157, Ser239, and Thr278—by PKA and PKG. These sites are phosphorylated with distinct kinetics both in vitro and in human platelets, contributing to VASP’s regulatory functions in cytoskeletal organization and platelet activity.

Background

Vasodilator-stimulated phosphoprotein (VASP) is an actin- and profilin-binding protein highly expressed in platelets, where it plays a key role in suppressing secretory and adhesive events. VASP is a major substrate for protein kinases regulated by cAMP and cGMP, such as protein kinase A (PKA) and protein kinase G (PKG). Additionally, it is directly phosphorylated at Ser157 by protein kinase C (PKC). Originally identified in platelets, VASP is phosphorylated in response to vasodilators like prostaglandin I2 (PGI2) and nitric oxide (NO), which increase intracellular cAMP and cGMP levels. Ena/VASP family proteins, including VASP, share a conserved structure featuring an N-terminal EVH1 (Ena/VASP-homology-1) domain, a central proline-rich region, and a C-terminal EVH2 domain. In vivo studies indicate that VASP negatively regulates platelet function. Ena/VASP proteins are essential for cell motility, migration, and adhesion. VASP contributes to profilin recruitment, actin nucleation, bundling, and filament formation, and it is thought to have anti-branching and anti-capping roles in actin dynamics. VASP localizes to key cytoskeletal and adhesive structures, including the leading edge of lamellipodia, actin stress fibers, filopodial tips, and focal adhesions, such as those involving the integrin αIIbβ3 in platelets. Phosphorylation of VASP occurs at three specific sites—Ser157, Ser239, and Thr278—by PKA and PKG. These sites are phosphorylated with distinct kinetics both in vitro and in human platelets, contributing to VASP’s regulatory functions in cytoskeletal organization and platelet activity.

References
[1] Wentworth JKT, et al. Biochem J. 2006 Jan 15;393(Pt 2):555-64.

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
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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%