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특이성	YTHDF1 Antibody [H2N8]는 YTHDF1 단백질의 총 내인성 수준을 검출합니다.
배경	YTHDF1은 단백질 번역, 줄기세포 자가 재생 및 배아 발달에 중요한 잘 연구된 m6A 리더 단백질입니다. 이는 단백질 번역 향상 및 mRNA 안정성 조절을 포함한 여러 메커니즘을 통해 표적 유전자의 발현을 조절합니다. YTHDF1의 세포 내 풍부도는 전사, 전사 후 및 번역 후 조절을 포함하는 복잡한 네트워크에 의해 엄격하게 제어됩니다. YTHDF1은 가장 널리 퍼진 m6A 리더로서, 특히 단백질 번역에서 m6A 변형 mRNA를 하류 운명과 연결합니다. 이는 eIF3b 번역 개시 복합체의 형성을 촉진하여 YAP와 같은 주요 표적의 번역을 촉진합니다. 이 과정은 eIF3b 고갈, YTHDF1 억제 또는 METTL3 매개 번역 이벤트 방해를 통해 방해될 수 있습니다. 또한 YTHDF1은 RNA 안정성에 중요한 역할을 하며, METTL3 촉매 m6A 변형에 의해 구동되는 효과인 c-Myc mRNA의 안정성과 발현을 증가시킵니다. 다른 YTH 계열 단백질도 YTHDC1이 주로 RNA 스플라이싱 및 핵 수출에서 기능하며 주로 핵에 국한되는 반면, YTHDC2는 특히 처리체 내에서 m6A 변형 mRNA 번역 및 분해에 필수적이며 YTHDF3은 YTHDF1과 협력하여 mRNA 번역을 향상시키고 YTHDF2를 통해 m6A 변형 전사체의 분해를 촉진하는 등 뚜렷한 역할을 보입니다. 따라서 이러한 단백질은 RNA 조절에서 m6A 리더의 다면적인 역할을 강조하며, YTHDF1은 배아 발달, 줄기세포 유지 및 암 진행의 핵심적인 역할을 합니다.

특이성

YTHDF1 Antibody [H2N8]는 YTHDF1 단백질의 총 내인성 수준을 검출합니다.

배경

YTHDF1은 단백질 번역, 줄기세포 자가 재생 및 배아 발달에 중요한 잘 연구된 m6A 리더 단백질입니다. 이는 단백질 번역 향상 및 mRNA 안정성 조절을 포함한 여러 메커니즘을 통해 표적 유전자의 발현을 조절합니다. YTHDF1의 세포 내 풍부도는 전사, 전사 후 및 번역 후 조절을 포함하는 복잡한 네트워크에 의해 엄격하게 제어됩니다. YTHDF1은 가장 널리 퍼진 m6A 리더로서, 특히 단백질 번역에서 m6A 변형 mRNA를 하류 운명과 연결합니다. 이는 eIF3b 번역 개시 복합체의 형성을 촉진하여 YAP와 같은 주요 표적의 번역을 촉진합니다. 이 과정은 eIF3b 고갈, YTHDF1 억제 또는 METTL3 매개 번역 이벤트 방해를 통해 방해될 수 있습니다. 또한 YTHDF1은 RNA 안정성에 중요한 역할을 하며, METTL3 촉매 m6A 변형에 의해 구동되는 효과인 c-Myc mRNA의 안정성과 발현을 증가시킵니다. 다른 YTH 계열 단백질도 YTHDC1이 주로 RNA 스플라이싱 및 핵 수출에서 기능하며 주로 핵에 국한되는 반면, YTHDC2는 특히 처리체 내에서 m6A 변형 mRNA 번역 및 분해에 필수적이며 YTHDF3은 YTHDF1과 협력하여 mRNA 번역을 향상시키고 YTHDF2를 통해 m6A 변형 전사체의 분해를 촉진하는 등 뚜렷한 역할을 보입니다. 따라서 이러한 단백질은 RNA 조절에서 m6A 리더의 다면적인 역할을 강조하며, YTHDF1은 배아 발달, 줄기세포 유지 및 암 진행의 핵심적인 역할을 합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:30

반응성

Human, Mouse, Rat

출처

Rabbit Monoclonal Antibody

MW

61 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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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 1234. 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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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

1234. 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://biomarkerres.biomedcentral.com/articles/10.1186/s40364-023-00452-1

적용 데이터

WB

Selleck 검증

Lane 1: HeLa
Lane 2: 293T
Lane 3: NIH/3T3
Lane 4: PC-12