데이터시트

생물학적 설명

특이성	Pan-TEAD Antibody [L14N23]는 총 TEAD 단백질의 내인성 수준을 인식합니다. 이 항체는 형질전환된 세포 추출물에서 TEAD 1, 2, 3 및 4를 인식하는 것으로 나타났습니다.
배경	Transcriptional Enhanced Associate Domain (TEAD) 전사 인자는 발생, 세포 증식, 조직 재생 및 조직 균형 유지 등 다양한 생물학적 과정에서 중추적인 역할을 합니다. 이들은 Hippo, Wnt, TGFβ 및 EGFR과 같은 여러 경로의 신호를 통합하고 조율하는 중심 허브 역할을 합니다. TEAD의 조절 이상은 KRAS, BRAF, LKB1, NF2 및 MYC와 같은 여러 잘 알려진 암 관련 유전자에 영향을 미쳐 종양 진행, 전이, 대사, 면역 반응 및 약물 저항성에 영향을 미칩니다. TEAD 패밀리는 TEAD1 (TEF-1/NTEF), TEAD2 (TEF-4/ETF), TEAD3 (TEF-5/ETFR-1), TEAD4 (TEF-3/ETFR-2/FR-19)의 4가지 진화적으로 보존된 전사 인자로 구성되며, 이들은 다양한 인간 조직에서 널리 발현됩니다. TEAD 패밀리의 각 구성원은 배아 발달 동안 심장 형성, 신경 발달 및 영양막 계통 결정과 같은 과정에 기여하며 조직 특이적 기능을 나타냅니다. 또한 TEAD 인자는 세포 증식 조절 및 세포 생물학에서 접촉 억제 시행에 중요합니다.

특이성

Pan-TEAD Antibody [L14N23]는 총 TEAD 단백질의 내인성 수준을 인식합니다. 이 항체는 형질전환된 세포 추출물에서 TEAD 1, 2, 3 및 4를 인식하는 것으로 나타났습니다.

배경

Transcriptional Enhanced Associate Domain (TEAD) 전사 인자는 발생, 세포 증식, 조직 재생 및 조직 균형 유지 등 다양한 생물학적 과정에서 중추적인 역할을 합니다. 이들은 Hippo, Wnt, TGFβ 및 EGFR과 같은 여러 경로의 신호를 통합하고 조율하는 중심 허브 역할을 합니다. TEAD의 조절 이상은 KRAS, BRAF, LKB1, NF2 및 MYC와 같은 여러 잘 알려진 암 관련 유전자에 영향을 미쳐 종양 진행, 전이, 대사, 면역 반응 및 약물 저항성에 영향을 미칩니다. TEAD 패밀리는 TEAD1 (TEF-1/NTEF), TEAD2 (TEF-4/ETF), TEAD3 (TEF-5/ETFR-1), TEAD4 (TEF-3/ETFR-2/FR-19)의 4가지 진화적으로 보존된 전사 인자로 구성되며, 이들은 다양한 인간 조직에서 널리 발현됩니다. TEAD 패밀리의 각 구성원은 배아 발달 동안 심장 형성, 신경 발달 및 영양막 계통 결정과 같은 과정에 기여하며 조직 특이적 기능을 나타냅니다. 또한 TEAD 인자는 세포 증식 조절 및 세포 생물학에서 접촉 억제 시행에 중요합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:100

반응성

Human, Mouse, Rat, Monkey

출처

Rabbit Monoclonal Antibody

MW

50 kDa, 53 kDa, 55 kDa, 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/Nuclear 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/Nuclear 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/Nuclear 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, 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 299. 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 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/Nuclear 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/Nuclear 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, 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

299. 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/31212916/

적용 데이터

WB

Selleck 검증

Lane 1: T-47D
Lane 2: ACHN
Lane 3: COS-7
Lane 4: HepG2
Lane 5: Vero
Lane 6: 3T3
Lane 7: F9