데이터시트

생물학적 설명

특이성	PAN TEAD Antibody [A12P9]는 총 Pan-TEAD 단백질의 내인성 수준을 감지합니다.
배경	TEAD1, TEAD2, TEAD3 및 TEAD4로 구성된 TEAD 전사 인자 계열은 세포 증식, 분화, 생존, 세포자멸사, 조직 성장 및 재생을 제어하는 유전자를 조절하는 보존된 DNA 결합 단백질 그룹입니다. 이들은 Hippo 신호 경로의 중심 하류 효과기로 작용하여 기계적 및 생화학적 입력을 통합하여 조직 항상성을 유지하고 장기 크기를 제어합니다. 모든 TEAD 단백질은 표적 프로모터에서 MCAT 요소를 인식하는 헬릭스-턴-헬릭스 접힘을 가진 N-말단 TEA DNA-결합 도메인(약 70~75개 아미노산)과 전사 보조 활성인자 YAP 및 TAZ를 모집하는 면역글로불린 유사 접힘을 가진 C-말단 YAP-결합 도메인(YBD)을 공유합니다. 이러한 보조 활성인자는 TEAD 단독으로는 고유 활성이 낮기 때문에 CTGF 및 CYR61과 같은 표적 유전자의 강력한 전사 활성화에 필수적입니다. TEAD 기능의 조정은 세포골격 역학, 상처 치유 및 조직 재생에 영향을 미칩니다. TEAD 계열 구성원의 조절 이상은 종종 비정상적인 YAP/TAZ 활성화를 통해 암 유발 전사 프로그램을 유도하고 전이를 촉진하며 발달 장애, 섬유증식성 질환 및 심근병증과 관련이 있습니다.

특이성

PAN TEAD Antibody [A12P9]는 총 Pan-TEAD 단백질의 내인성 수준을 감지합니다.

배경

TEAD1, TEAD2, TEAD3 및 TEAD4로 구성된 TEAD 전사 인자 계열은 세포 증식, 분화, 생존, 세포자멸사, 조직 성장 및 재생을 제어하는 유전자를 조절하는 보존된 DNA 결합 단백질 그룹입니다. 이들은 Hippo 신호 경로의 중심 하류 효과기로 작용하여 기계적 및 생화학적 입력을 통합하여 조직 항상성을 유지하고 장기 크기를 제어합니다. 모든 TEAD 단백질은 표적 프로모터에서 MCAT 요소를 인식하는 헬릭스-턴-헬릭스 접힘을 가진 N-말단 TEA DNA-결합 도메인(약 70~75개 아미노산)과 전사 보조 활성인자 YAP 및 TAZ를 모집하는 면역글로불린 유사 접힘을 가진 C-말단 YAP-결합 도메인(YBD)을 공유합니다. 이러한 보조 활성인자는 TEAD 단독으로는 고유 활성이 낮기 때문에 CTGF 및 CYR61과 같은 표적 유전자의 강력한 전사 활성화에 필수적입니다. TEAD 기능의 조정은 세포골격 역학, 상처 치유 및 조직 재생에 영향을 미칩니다. TEAD 계열 구성원의 조절 이상은 종종 비정상적인 YAP/TAZ 활성화를 통해 암 유발 전사 프로그램을 유도하고 전이를 촉진하며 발달 장애, 섬유증식성 질환 및 심근병증과 관련이 있습니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

48 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 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 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 1. 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/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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. Lyse the cells by adding an appropriate volume of RIPA/Nuclear Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

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 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

1. 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/18579750/
https://pubmed.ncbi.nlm.nih.gov/20123908/

적용 데이터

WB

Selleck 검증

Lane 1: HeLa, Lane 2: SW480