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생물학적 설명

특이성	Phospho-Akt (Thr450) Antibody [J1B12]는 Thr450에서 인산화된 경우에만 내인성 Phospho-Akt 수준을 인식합니다.
배경	PKB 또는 Rac으로도 알려진 Akt는 세포 생존 및 세포자멸사를 조절하는 중요한 단백질 키나아제입니다. 인슐린 및 다양한 성장 인자에 의해 워트만닌에 민감한 PI3 키나아제 의존적 경로를 통해 활성화됩니다. Akt의 활성화는 인지질에 대한 결합과 PDK1에 의한 활성화 루프 내 Thr308에서의 후속 인산화, 그리고 C-말단 영역의 Ser473에서의 인산화를 필요로 합니다. Akt는 Bad, forkhead 전사 인자, c-Raf 및 caspase-9를 포함한 주요 표적의 인산화 및 비활성화를 통해 세포자멸사를 억제하여 세포 생존을 향상시킵니다. PTEN 인산가수분해효소는 PI3K/Akt 신호 캐스케이드의 주요 음성 조절자로 작용합니다. Akt는 세포 생존 및 글리코겐 합성을 촉진하는 역할 외에도, GSK-3β 매개 Cyclin D1 인산화 및 분해를 방지하고 Cyclin 의존성 키나아제 억제제 p27 Kip1 및 p21 Waf1/Cip1을 하향 조절함으로써 세포 주기를 조절합니다. 허혈성 손상에 반응하여 Akt는 JNK에 의해 재활성화될 수 있으며, JNK는 Thr450을 인산화하여 3-phosphoinositide-dependent protein kinase-1에 의한 추가 인산화를 위한 중요한 프라이밍 단계가 되며 Akt의 활성을 회복시킵니다.

특이성

Phospho-Akt (Thr450) Antibody [J1B12]는 Thr450에서 인산화된 경우에만 내인성 Phospho-Akt 수준을 인식합니다.

배경

PKB 또는 Rac으로도 알려진 Akt는 세포 생존 및 세포자멸사를 조절하는 중요한 단백질 키나아제입니다. 인슐린 및 다양한 성장 인자에 의해 워트만닌에 민감한 PI3 키나아제 의존적 경로를 통해 활성화됩니다. Akt의 활성화는 인지질에 대한 결합과 PDK1에 의한 활성화 루프 내 Thr308에서의 후속 인산화, 그리고 C-말단 영역의 Ser473에서의 인산화를 필요로 합니다. Akt는 Bad, forkhead 전사 인자, c-Raf 및 caspase-9를 포함한 주요 표적의 인산화 및 비활성화를 통해 세포자멸사를 억제하여 세포 생존을 향상시킵니다. PTEN 인산가수분해효소는 PI3K/Akt 신호 캐스케이드의 주요 음성 조절자로 작용합니다. Akt는 세포 생존 및 글리코겐 합성을 촉진하는 역할 외에도, GSK-3β 매개 Cyclin D1 인산화 및 분해를 방지하고 Cyclin 의존성 키나아제 억제제 p27 Kip1 및 p21 Waf1/Cip1을 하향 조절함으로써 세포 주기를 조절합니다. 허혈성 손상에 반응하여 Akt는 JNK에 의해 재활성화될 수 있으며, JNK는 Thr450을 인산화하여 3-phosphoinositide-dependent protein kinase-1에 의한 추가 인산화를 위한 중요한 프라이밍 단계가 되며 Akt의 활성을 회복시킵니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

60 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, 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 811. 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, 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

811. 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/16962653/
https://pubmed.ncbi.nlm.nih.gov/16306447/

적용 데이터

WB

Selleck 검증

Lane 1: LNCaP
Lane 2: LNCaP (serum-starved; Torin, 10.5 µM, 5h)
Lane 3: LNCaP (Torin, 10.5 µM, 5h)
Lane 4: LNCaP (Torin, 0.5 µM, 24h)