Phospho-PDGF Receptor β (Tyr771) Antibody [C22C4]

Catalog No.: F1497

Application: Reactivity: Mouse

Lane 1: NIH/3T3
Lane 2: NIH/3T3 (PDGF-BB-treated)

サイズ（液体）	価格(税別)	在庫状況
20uL	JPY 20000	国内在庫なし(納期7~10日)
100uL	JPY 64800	国内在庫あり
100uL*2	JPY 97200	お問い合わせ

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キーポイント

WB
SDS-PAGE の分離ゲルの推奨濃度：5%。

使用情報

Dilution
WB1:1000

Application
WB

Source
Rabbit Monoclonal Antibody

Reactivity
Mouse

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
190 kDa

ポジティブコントロール	NIH/3T3 (treated with PDGF-BB, 100 ng/mL, 5 min)
ネガティブコントロール	NIH/3T3

サンプル処理データの例

サンプル	処理状況
NIH/3T3	PDGF-BB-treated (100 ng/mL, 5 min)
クリックして、さらに多くのサンプルデータを表示

^*異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

サンプル	処理状況

プロトコール

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: 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 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 1285. 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: 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 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

1285. 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

生物学的記述

Specificity
Phospho-PDGF Receptor β (Tyr771) Antibody [C22C4] detects endogenous levels of PDGF receptor β only when phosphorylated at tyrosine 771.

タンパク質の局在
細胞膜、細胞質小胞、リソソーム、細胞内膜系

Uniprot ID
P09619

Clone
C22C4

Background
The platelet-derived growth factor (PDGF) family consists of several dimeric isoforms linked by disulfide bonds, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. These isoforms exhibit specific binding affinities to two closely related receptor tyrosine kinases: PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα homodimers interact with all PDGF isoforms except those containing PDGF-D, whereas PDGFRβ homodimers bind to PDGF-BB, PDGF-DD, and the PDGF-AB heterodimer. The heterodimeric receptor α/β recognizes PDGF-B, PDGF-C, and PDGF-D homodimers, as well as the PDGF-AB heterodimer. Ligand binding triggers receptor dimerization and autophosphorylation, enabling the recruitment and activation of cytoplasmic signaling proteins with SH2 domains, such as GRB2, Src, GAP, PI3K, PLCγ, and NCK. These activated receptors initiate signaling pathways that regulate essential cellular functions, including proliferation, cytoskeletal reorganization, migration, and differentiation. Tyrosine 771 on the PDGF β receptor serves as a docking site for Ras GTPase-activating protein (RasGAP). SHP-2 has been shown to play a critical role in regulating the phosphorylation of tyrosine 771, thereby influencing RasGAP recruitment and modulating Ras/MAPK signaling pathways mediated by PDGF receptors.

Background

The platelet-derived growth factor (PDGF) family consists of several dimeric isoforms linked by disulfide bonds, including PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. These isoforms exhibit specific binding affinities to two closely related receptor tyrosine kinases: PDGF receptor α (PDGFRα) and PDGF receptor β (PDGFRβ). PDGFRα homodimers interact with all PDGF isoforms except those containing PDGF-D, whereas PDGFRβ homodimers bind to PDGF-BB, PDGF-DD, and the PDGF-AB heterodimer. The heterodimeric receptor α/β recognizes PDGF-B, PDGF-C, and PDGF-D homodimers, as well as the PDGF-AB heterodimer. Ligand binding triggers receptor dimerization and autophosphorylation, enabling the recruitment and activation of cytoplasmic signaling proteins with SH2 domains, such as GRB2, Src, GAP, PI3K, PLCγ, and NCK. These activated receptors initiate signaling pathways that regulate essential cellular functions, including proliferation, cytoskeletal reorganization, migration, and differentiation. Tyrosine 771 on the PDGF β receptor serves as a docking site for Ras GTPase-activating protein (RasGAP). SHP-2 has been shown to play a critical role in regulating the phosphorylation of tyrosine 771, thereby influencing RasGAP recruitment and modulating Ras/MAPK signaling pathways mediated by PDGF receptors.

References
[1] Bergsten E, et al. Nat Cell Biol. 2001 May;3(5):512-6. [2] Kashishian A, et al. EMBO J. 1992 Apr;11(4):1373-82.

PVDFメンブレン孔径参考表

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%