데이터시트

생물학적 설명

특이성	Phospho-Akt2 (Ser474) Antibody [L16H11]는 Ser474에서 인산화된 경우에만 내인성 Akt2 단백질 수준을 인식합니다.
배경	Akt는 Protein Kinase B (PKB)라고도 알려져 있으며, S6K, RSK, SGK 및 PKC와 같은 다른 키나아제를 포함하는 AGC 키나아제 계열에 속합니다. Akt를 포함한 이 계열의 공통적인 특징은 두 가지 중요한 부위에서 인산화된다는 것입니다: 촉매 부위(활성화 루프 또는 T-루프)와 C-말단 소수성 모티프(HM) 부위. 세린-트레오닌 키나아제인 Akt2는 Akt/PKB 원발암유전자 계열의 일부입니다. Akt1과 Akt2 모두 포스파티딜이노시톨 3-키나아제 (PI 3-키나아제)의 하류에서 신호 변환기로 작용합니다. 별개의 유전자에 의해 코딩됨에도 불구하고, Akt1과 Akt2는 카르복시-말단 영역에서 가장 큰 차이를 보입니다. Akt1은 글리코겐 대사 조절뿐만 아니라 세포 성장 및 생존에 중요한 역할을 합니다. 포유류 라파마이신 표적 (mTOR)은 랩터-mTOR (TORC1) 및 릭터-mTOR (TORC2)의 두 가지 복합체를 통해 세포 성장 및 증식에 영향을 미칩니다. TORC2는 소수성 모티프에서 Ser473의 Akt/PKB 인산화를 담당하는 찾기 어려운 PDK2로 확인되었습니다. 이 인산화는 활성화 루프의 Thr308과 함께 Akt의 적절한 기능에 필수적입니다.

특이성

Phospho-Akt2 (Ser474) Antibody [L16H11]는 Ser474에서 인산화된 경우에만 내인성 Akt2 단백질 수준을 인식합니다.

배경

Akt는 Protein Kinase B (PKB)라고도 알려져 있으며, S6K, RSK, SGK 및 PKC와 같은 다른 키나아제를 포함하는 AGC 키나아제 계열에 속합니다. Akt를 포함한 이 계열의 공통적인 특징은 두 가지 중요한 부위에서 인산화된다는 것입니다: 촉매 부위(활성화 루프 또는 T-루프)와 C-말단 소수성 모티프(HM) 부위. 세린-트레오닌 키나아제인 Akt2는 Akt/PKB 원발암유전자 계열의 일부입니다. Akt1과 Akt2 모두 포스파티딜이노시톨 3-키나아제 (PI 3-키나아제)의 하류에서 신호 변환기로 작용합니다. 별개의 유전자에 의해 코딩됨에도 불구하고, Akt1과 Akt2는 카르복시-말단 영역에서 가장 큰 차이를 보입니다. Akt1은 글리코겐 대사 조절뿐만 아니라 세포 성장 및 생존에 중요한 역할을 합니다. 포유류 라파마이신 표적 (mTOR)은 랩터-mTOR (TORC1) 및 릭터-mTOR (TORC2)의 두 가지 복합체를 통해 세포 성장 및 증식에 영향을 미칩니다. TORC2는 소수성 모티프에서 Ser473의 Akt/PKB 인산화를 담당하는 찾기 어려운 PDK2로 확인되었습니다. 이 인산화는 활성화 루프의 Thr308과 함께 Akt의 적절한 기능에 필수적입니다.

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 1010. 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

1010. 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/11753568/
https://pubmed.ncbi.nlm.nih.gov/16962653/

적용 데이터

WB

Selleck 검증

Lane 1: MEF (KO Akt1)
Lane 2: MEF (KO Akt1; hPDGF-AA, 100 ng/ml, 15 min)
Lane 3: MEF (KO Akt2)
Lane 4: MEF (KO Akt2; hPDGF-AA, 100 ng/ml, 15 min)