데이터시트

생물학적 설명

특이성	14-3-3 γ Antibody [J16F21]는 총 14-3-3 γ 단백질의 내인성 수준을 인식합니다. 이 항체는 다른 포유류 14-3-3 이소폼과 교차 반응하지 않으며, 개구리와 물고기를 포함한 다른 종의 14-3-3γ 오르토로그를 검출할 것으로 예측됩니다.
배경	14-3-3γ는 풍부한 뇌 단백질이며, 247개의 아미노산으로 구성된 14-3-3 계열의 7가지 이소폼 중 하나입니다. 각 프로토머가 9개의 역평행 α-나선을 포함하는 이량체 구조를 특징으로 합니다. 14-3-3γ는 다양한 표적 단백질의 인산화된 세린/트레오닌 모티프에 결합하여, 스캐폴드로 작용하여 그들의 활동, 위치, 안정성 또는 상호작용을 조절합니다. 이는 신호 전달, Cell Cycle 진행, 세포 사멸, 스트레스 반응 및 대사와 같은 중요한 세포 과정에 관여합니다. 14-3-3γ는 14-3-3ε와 같은 다른 이소폼과 이종 이합체를 형성하여 상피 나트륨 채널(ENaC)과 같은 특정 표적에 선택적으로 결합할 수 있습니다. Ser58에서의 인산화는 그 이합체화와 표적 결합에 영향을 미칩니다. 14-3-3γ는 KSR1을 탈인산화로부터 보호하여 ERK2 활성화를 유지하고, Ca2+/CaM 결합을 억제하면서 DAPK2 이합체화를 안정화시켜 세포 신호 전달 및 스트레스 반응에 영향을 미칩니다. 14-3-3γ는 Chk1 및 Cdc25A를 모집하는 스캐폴드로 작용하여 S76에서 Chk1 매개 Cdc25A 인산화를 촉진하며, 이는 그 후 폴리유비퀴틴화 및 프로테아좀 의존적 분해로 이어집니다.

특이성

14-3-3 γ Antibody [J16F21]는 총 14-3-3 γ 단백질의 내인성 수준을 인식합니다. 이 항체는 다른 포유류 14-3-3 이소폼과 교차 반응하지 않으며, 개구리와 물고기를 포함한 다른 종의 14-3-3γ 오르토로그를 검출할 것으로 예측됩니다.

배경

14-3-3γ는 풍부한 뇌 단백질이며, 247개의 아미노산으로 구성된 14-3-3 계열의 7가지 이소폼 중 하나입니다. 각 프로토머가 9개의 역평행 α-나선을 포함하는 이량체 구조를 특징으로 합니다. 14-3-3γ는 다양한 표적 단백질의 인산화된 세린/트레오닌 모티프에 결합하여, 스캐폴드로 작용하여 그들의 활동, 위치, 안정성 또는 상호작용을 조절합니다. 이는 신호 전달, Cell Cycle 진행, 세포 사멸, 스트레스 반응 및 대사와 같은 중요한 세포 과정에 관여합니다. 14-3-3γ는 14-3-3ε와 같은 다른 이소폼과 이종 이합체를 형성하여 상피 나트륨 채널(ENaC)과 같은 특정 표적에 선택적으로 결합할 수 있습니다. Ser58에서의 인산화는 그 이합체화와 표적 결합에 영향을 미칩니다. 14-3-3γ는 KSR1을 탈인산화로부터 보호하여 ERK2 활성화를 유지하고, Ca2+/CaM 결합을 억제하면서 DAPK2 이합체화를 안정화시켜 세포 신호 전달 및 스트레스 반응에 영향을 미칩니다. 14-3-3γ는 Chk1 및 Cdc25A를 모집하는 스캐폴드로 작용하여 S76에서 Chk1 매개 Cdc25A 인산화를 촉진하며, 이는 그 후 폴리유비퀴틴화 및 프로테아좀 의존적 분해로 이어집니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human, Mouse, Rat, Monkey, Pig, Chicken, Xenopus, Zebrafish

출처

Rabbit Monoclonal Antibody

MW

27 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 1:1000), 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 863. 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 1:1000), 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

863. 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/
https://pubmed.ncbi.nlm.nih.gov/29915393/

적용 데이터

WB

Selleck 검증

Lane 1: Hela
Lane 2: A431
Lane 3: COS7
Lane 4: 3T3

IHC

Selleck 검증

Immunohistochemical analysis of formalin fixed paraffin embedded human breast cancer tissue with F1473 at 1/100 dilution.