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생물학적 설명

특이성	Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody [L15L21]는 Tyr319에서 인산화될 때에만 내인성 Zap-70 수준을 검출합니다. Tyr352에서 인산화될 때 내인성 Syk 수준과 교차 반응합니다.
배경	Protein Tyrosine Kinase인 Syk 계열에 속하는 ZAP-70은 T 및 NK 세포에서 발현되며, 항원 수용체 결합을 통해 T 세포 반응을 시작하는 핵심적인 역할을 합니다. 이 세포질 키나제는 T 세포 수용체(TCR) 활성화에 의해 유발되는 신호 전달 캐스케이드에 중요합니다. 구조적으로 ZAP-70은 두 개의 SH2 도메인과 카르복시 말단 키나제 도메인으로 구성됩니다. TCR 자극 시, ZAP-70 내의 여러 티로신 잔기는 Src 계열의 티로신 키나제 Lck에 의해 인산화됩니다. 이러한 인산화 이벤트는 ZAP-70의 조절 및 활성, 그리고 다른 신호 분자와의 상호작용에 필수적입니다. 티로신 잔기 Y292, Y315 및 Y319는 ZAP-70의 도메인 간 B 영역 내에 위치합니다. 인산화될 때, 이 잔기들은 하류 신호 분자를 위한 도킹 부위 역할을 하는 것으로 여겨집니다. 특히, Y315와 Y319를 모두 페닐알라닌으로 돌연변이시키면 키나제 활성 손실이 발생하여 ZAP-70 기능에서 이들의 중요성을 강조합니다.

특이성

Phospho-Zap-70 (Tyr319)/Syk (Tyr352) Antibody [L15L21]는 Tyr319에서 인산화될 때에만 내인성 Zap-70 수준을 검출합니다. Tyr352에서 인산화될 때 내인성 Syk 수준과 교차 반응합니다.

배경

Protein Tyrosine Kinase인 Syk 계열에 속하는 ZAP-70은 T 및 NK 세포에서 발현되며, 항원 수용체 결합을 통해 T 세포 반응을 시작하는 핵심적인 역할을 합니다. 이 세포질 키나제는 T 세포 수용체(TCR) 활성화에 의해 유발되는 신호 전달 캐스케이드에 중요합니다. 구조적으로 ZAP-70은 두 개의 SH2 도메인과 카르복시 말단 키나제 도메인으로 구성됩니다. TCR 자극 시, ZAP-70 내의 여러 티로신 잔기는 Src 계열의 티로신 키나제 Lck에 의해 인산화됩니다. 이러한 인산화 이벤트는 ZAP-70의 조절 및 활성, 그리고 다른 신호 분자와의 상호작용에 필수적입니다. 티로신 잔기 Y292, Y315 및 Y319는 ZAP-70의 도메인 간 B 영역 내에 위치합니다. 인산화될 때, 이 잔기들은 하류 신호 분자를 위한 도킹 부위 역할을 하는 것으로 여겨집니다. 특히, Y315와 Y319를 모두 페닐알라닌으로 돌연변이시키면 키나제 활성 손실이 발생하여 ZAP-70 기능에서 이들의 중요성을 강조합니다.

사용 정보

응용

WB, IF, FCM

희석

WB	IF	FCM
1:1000	1:200 - 1:800	1:200 - 1:800

반응성

Human, Mouse

출처

Rabbit Monoclonal Antibody

MW

70, 72 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 1011. 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.
IF	Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

참조

https://pubmed.ncbi.nlm.nih.gov/20452964/

적용 데이터

WB

Selleck 검증

Lane 1: Jurkat (hydrogen peroxide, 2mM, 2min)
Lane 2: Jurkat (λ phosphatase treated)

IF

Selleck 검증

Immunofluorescent analysis of Jurkat cells using F0556 (green, 1:200), Hoechst (blue) and tubulin (Red).