데이터시트

생물학적 설명

특이성	Phospho-Zyxin (Ser142/143) Antibody [L24L6]는 Ser142 및 Ser143에서 인산화된 경우에만 내인성 지신 단백질 수준을 인식합니다.
배경	인산화-지신 (Ser 142/143)은 지신의 인산화된 형태로, 액틴 세포골격 조직 및 세포 신호 전달에 중요한 기계감수성 LIM 도메인 함유 초점 접착 단백질입니다. 다양한 세포 유형, 특히 내피세포 및 근육세포에서 발현되며, PKA와 같은 키나아제에 의해 매개되는 세린 잔기 142 및 143에서의 인산화는 액틴 필라멘트 및 초점 접착 구성 요소와의 상호작용을 강화합니다. 구조적으로 지신은 프롤린이 풍부한 ActA 영역, LIM 도메인 및 이러한 인산화 부위를 포함하는 N-말단 영역을 포함하며, 이는 기능을 조절하기 위한 구조적 변화를 유도합니다. 인산화-지신은 내피세포 외포작용, 특히 혈관 무결성을 위한 폰 빌레브란트 인자 분비에서 필수적인 역할을 하며, 세포 형태, 운동성 및 기계적 안정성에 중요한 액틴 세포골격 재형성에서도 중요한 역할을 합니다. 이러한 인산화 이벤트는 세포외 신호를 통합하고 세포골격 역학을 조절하는 데 필수적이며, 인산화-지신을 혈관 생물학 및 세포 반응의 핵심 요소로 만듭니다.

특이성

Phospho-Zyxin (Ser142/143) Antibody [L24L6]는 Ser142 및 Ser143에서 인산화된 경우에만 내인성 지신 단백질 수준을 인식합니다.

배경

인산화-지신 (Ser 142/143)은 지신의 인산화된 형태로, 액틴 세포골격 조직 및 세포 신호 전달에 중요한 기계감수성 LIM 도메인 함유 초점 접착 단백질입니다. 다양한 세포 유형, 특히 내피세포 및 근육세포에서 발현되며, PKA와 같은 키나아제에 의해 매개되는 세린 잔기 142 및 143에서의 인산화는 액틴 필라멘트 및 초점 접착 구성 요소와의 상호작용을 강화합니다. 구조적으로 지신은 프롤린이 풍부한 ActA 영역, LIM 도메인 및 이러한 인산화 부위를 포함하는 N-말단 영역을 포함하며, 이는 기능을 조절하기 위한 구조적 변화를 유도합니다. 인산화-지신은 내피세포 외포작용, 특히 혈관 무결성을 위한 폰 빌레브란트 인자 분비에서 필수적인 역할을 하며, 세포 형태, 운동성 및 기계적 안정성에 중요한 액틴 세포골격 재형성에서도 중요한 역할을 합니다. 이러한 인산화 이벤트는 세포외 신호를 통합하고 세포골격 역학을 조절하는 데 필수적이며, 인산화-지신을 혈관 생물학 및 세포 반응의 핵심 요소로 만듭니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:200

반응성

Human, Mouse, Rat, Monkey, Dog

출처

Rabbit Monoclonal Antibody

MW

78 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 1363. 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. (Exposure time of at least 90s is recommended)

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

1363. 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. (Exposure time of at least 90s is recommended)

참조

https://pubmed.ncbi.nlm.nih.gov/33572997/
https://pubmed.ncbi.nlm.nih.gov/28256511/

적용 데이터

WB

Selleck 검증

Lane 1: A-431
Lane 2: A-431 (λ/CIP treated)
Lane 3: U-2 OS
Lane 4: U-2 OS (λ/CIP treated)