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특이성	Phospho-(Ser) 14-3-3 Binding Motif Antibody [P9J15]는 세린 잔기에서 인산화된 경우에만 14-3-3 결합 모티프의 내인성 수준을 인식합니다.
배경	Phospho-(Ser) 14-3-3 결합 모티프는 표적 단백질에서 발견되는 핵심 서열로, 세린 잔기에서 인산화될 때 14-3-3 단백질과 선택적으로 상호작용합니다. 이 인산화는 14-3-3 결합을 활성화하는 분자 스위치 역할을 하며, 표적 단백질의 기능적 영역 근처에 위치합니다. 일단 결합하면 14-3-3 이량체는 단백질을 안정화시켜 위치, 활동 또는 안정성에 영향을 미칩니다. 이 결합의 결과는 다양하며, 세포질 내 단백질 격리, 효소 기능 억제 또는 다른 분자와의 상호작용 차단 등을 포함할 수 있습니다. 세포 주기 조절 중 Cdc25B 및 DAPK2와 같은 단백질의 특정 부위 인산화는 14-3-3 결합을 유도하여 기능을 억제하거나 세포질로 이동시킵니다. 이 상호작용은 또한 인산화 부위를 탈인산화로부터 보호하여 표적 단백질이 필요에 따라 기능을 유지하거나 비활성 상태를 유지하도록 할 수 있습니다. 14-3-3 결합은 또한 단백질을 분해로부터 보호하거나 적절한 신호 전달에 필수적인 다중 단백질 복합체의 조립을 촉진함으로써 단백질-단백질 상호작용에서 중요한 역할을 합니다.

특이성

Phospho-(Ser) 14-3-3 Binding Motif Antibody [P9J15]는 세린 잔기에서 인산화된 경우에만 14-3-3 결합 모티프의 내인성 수준을 인식합니다.

배경

Phospho-(Ser) 14-3-3 결합 모티프는 표적 단백질에서 발견되는 핵심 서열로, 세린 잔기에서 인산화될 때 14-3-3 단백질과 선택적으로 상호작용합니다. 이 인산화는 14-3-3 결합을 활성화하는 분자 스위치 역할을 하며, 표적 단백질의 기능적 영역 근처에 위치합니다. 일단 결합하면 14-3-3 이량체는 단백질을 안정화시켜 위치, 활동 또는 안정성에 영향을 미칩니다. 이 결합의 결과는 다양하며, 세포질 내 단백질 격리, 효소 기능 억제 또는 다른 분자와의 상호작용 차단 등을 포함할 수 있습니다. 세포 주기 조절 중 Cdc25B 및 DAPK2와 같은 단백질의 특정 부위 인산화는 14-3-3 결합을 유도하여 기능을 억제하거나 세포질로 이동시킵니다. 이 상호작용은 또한 인산화 부위를 탈인산화로부터 보호하여 표적 단백질이 필요에 따라 기능을 유지하거나 비활성 상태를 유지하도록 할 수 있습니다. 14-3-3 결합은 또한 단백질을 분해로부터 보호하거나 적절한 신호 전달에 필수적인 다중 단백질 복합체의 조립을 촉진함으로써 단백질-단백질 상호작용에서 중요한 역할을 합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:4000	1:20

반응성

ALL

출처

Rabbit Monoclonal Antibody

MW

29 kDa

보관 완충액

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

보관
(수령일로부터)

-20°C (avoid freeze-thaw cycles), 2 years

실험 방법
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)，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), 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), 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, 60 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 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 1:4000), 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 1389. 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.

실험 방법

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)，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), 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), 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, 60 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 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 1:4000), 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

1389. 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.

참조

https://pubmed.ncbi.nlm.nih.gov/36188227/

적용 데이터

WB

Selleck 검증

Lane 1: A431, Lane 2: A431 (calyculin A, 100nM, 30min)