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특이성	TCF4/TCF7L2 Antibody [P1D24]는 총 TCF4/TCF7L2 단백질의 내인성 수준을 감지합니다.
배경	TCF4(전사 인자 4) 및 TCF7L2(T-세포 인자 7-유사 2)는 세포 운명, 분화 및 신진대사를 조절하는 Wnt/β-카테닌 신호 전달 경로의 중심인 TCF/LEF 계열의 전사 인자입니다. 구조적으로 두 단백질 모두 DNA 결합 및 전사 조절을 가능하게 하는 고이동성 그룹(HMG) 박스 도메인을 포함합니다. 이들은 Groucho/TLE과 같은 보조 억제자를 모집하여 β-카테닌이 없을 때 전사 억제자로 작용하지만, β-카테닌이 결합하면 활성인자로 전환되어 Wnt 표적 유전자의 발현을 촉진합니다. TCF4는 주로 신경 발달에 관여하여 신경 전구 세포 분화 및 성숙을 조절하며, TCF7L2는 지방 생성, 포도당 항상성 및 인슐린 분비에 영향을 미치는 대사 조절에 중요한 역할을 합니다. TCF4는 뇌 발달에 중요하며 TCF4 돌연변이로 인한 신경 발달 장애인 피트-홉킨스 증후군을 유발합니다. TCF7L2는 제2형 당뇨병(T2D)과 관련이 있으며, rs7903146과 같은 유전적 변이는 인슐린 분비 및 β-세포 기능을 손상시켜 질병 위험을 증가시킵니다. TCF4의 조절 이상은 신경학적 장애 및 대장암과 같은 암에 기여하는 반면, TCF7L2는 대사 질환 및 심혈관 건강과 관련이 있습니다.

특이성

TCF4/TCF7L2 Antibody [P1D24]는 총 TCF4/TCF7L2 단백질의 내인성 수준을 감지합니다.

배경

TCF4(전사 인자 4) 및 TCF7L2(T-세포 인자 7-유사 2)는 세포 운명, 분화 및 신진대사를 조절하는 Wnt/β-카테닌 신호 전달 경로의 중심인 TCF/LEF 계열의 전사 인자입니다. 구조적으로 두 단백질 모두 DNA 결합 및 전사 조절을 가능하게 하는 고이동성 그룹(HMG) 박스 도메인을 포함합니다. 이들은 Groucho/TLE과 같은 보조 억제자를 모집하여 β-카테닌이 없을 때 전사 억제자로 작용하지만, β-카테닌이 결합하면 활성인자로 전환되어 Wnt 표적 유전자의 발현을 촉진합니다. TCF4는 주로 신경 발달에 관여하여 신경 전구 세포 분화 및 성숙을 조절하며, TCF7L2는 지방 생성, 포도당 항상성 및 인슐린 분비에 영향을 미치는 대사 조절에 중요한 역할을 합니다. TCF4는 뇌 발달에 중요하며 TCF4 돌연변이로 인한 신경 발달 장애인 피트-홉킨스 증후군을 유발합니다. TCF7L2는 제2형 당뇨병(T2D)과 관련이 있으며, rs7903146과 같은 유전적 변이는 인슐린 분비 및 β-세포 기능을 손상시켜 질병 위험을 증가시킵니다. TCF4의 조절 이상은 신경학적 장애 및 대장암과 같은 암에 기여하는 반면, TCF7L2는 대사 질환 및 심혈관 건강과 관련이 있습니다.

사용 정보

응용

WB, IP, ChIP

희석

WB	IP	CHIP
1:1000	1:50	1:50

반응성

Human

출처

Rabbit Monoclonal Antibody

MW

58 kDa, 79 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 1399. 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

1399. 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/34016596/
https://pubmed.ncbi.nlm.nih.gov/34568447/

적용 데이터

WB

Selleck 검증

Lane 1: HCT116, Lane 2: SW620, Lane 3: HT15