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생물학적 설명

특이성	CNBP Antibody [E10D2]는 총 CNBP 단백질의 내인성 수준을 감지합니다.
배경	CNBP (ZNF9)는 배아 발달 및 전사 후 유전자 조절에 중요한 고도로 보존된 CCHC형 아연 핑거 핵산 결합 단백질입니다. 이 단백질은 7개의 탠덤 아연 너클 모티프를 특징으로 하며, 이 모티프는 아연 이온을 조절하여 컴팩트한 ββα 접힘을 형성하고, G-rich 단일 가닥 DNA 및 RNA 서열에 대한 고친화성 결합을 가능하게 합니다. 첫 번째와 두 번째 아연 핑거 사이에 RGG 상자가 존재하면 상 분리된 생체 분자 액적 형성이 촉진되고 협력적 핵산 리모델링이 향상됩니다. CNBP는 G-쿼드러플렉스(G4) 샤페론으로 기능하여 c-Myc 프로모터 내의 안정적인 G4 구조를 적극적으로 풀어 RNA 폴리머라제 II 전사를 촉진하고, ODC mRNA의 5'UTR 내에서 캡 비의존적 번역을 용이하게 합니다. CNBP는 직접적인 G4 의존적 프로모터 결합을 통해 β-카테닌 및 IL-6과 같은 유전자를 전사 활성화할 수 있으며, 세포 스트레스 및 사이토카인 신호에 대한 반응으로 인산화에 의해 유발되는 이합체화 및 핵 전위에 의해 그 활성과 국소화가 동적으로 조절됩니다. CNBP 인트론 1에서 (CCTG)n 반복 확장은 제2형 근긴장성 이영양증(DM2)의 원인이 되며, 돌연변이 RNA 전사체는 기능성 CNBP를 격리시켜 muscleblind 유사 단백질 독성, myf5 G4 안정화로 인한 근육 형성 장애 및 진행성 다기관 위축을 초래합니다.

특이성

CNBP Antibody [E10D2]는 총 CNBP 단백질의 내인성 수준을 감지합니다.

배경

CNBP (ZNF9)는 배아 발달 및 전사 후 유전자 조절에 중요한 고도로 보존된 CCHC형 아연 핑거 핵산 결합 단백질입니다. 이 단백질은 7개의 탠덤 아연 너클 모티프를 특징으로 하며, 이 모티프는 아연 이온을 조절하여 컴팩트한 ββα 접힘을 형성하고, G-rich 단일 가닥 DNA 및 RNA 서열에 대한 고친화성 결합을 가능하게 합니다. 첫 번째와 두 번째 아연 핑거 사이에 RGG 상자가 존재하면 상 분리된 생체 분자 액적 형성이 촉진되고 협력적 핵산 리모델링이 향상됩니다. CNBP는 G-쿼드러플렉스(G4) 샤페론으로 기능하여 c-Myc 프로모터 내의 안정적인 G4 구조를 적극적으로 풀어 RNA 폴리머라제 II 전사를 촉진하고, ODC mRNA의 5'UTR 내에서 캡 비의존적 번역을 용이하게 합니다. CNBP는 직접적인 G4 의존적 프로모터 결합을 통해 β-카테닌 및 IL-6과 같은 유전자를 전사 활성화할 수 있으며, 세포 스트레스 및 사이토카인 신호에 대한 반응으로 인산화에 의해 유발되는 이합체화 및 핵 전위에 의해 그 활성과 국소화가 동적으로 조절됩니다. CNBP 인트론 1에서 (CCTG)n 반복 확장은 제2형 근긴장성 이영양증(DM2)의 원인이 되며, 돌연변이 RNA 전사체는 기능성 CNBP를 격리시켜 muscleblind 유사 단백질 독성, myf5 G4 안정화로 인한 근육 형성 장애 및 진행성 다기관 위축을 초래합니다.

사용 정보

응용

WB, IP

희석

WB

1:5000-1:50000

반응성

Mouse, Rat, Human

출처

Mouse Monoclonal Antibody

MW

19 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)，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/NP-40 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/NP-40 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 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 1:5000), 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/NP-40 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/NP-40 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/NP-40 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. ( 0.22 µm PVDF membrane is recommended )） Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 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 1:5000), 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/31219592/
https://pubmed.ncbi.nlm.nih.gov/34474118/

적용 데이터

WB

Selleck 검증

Lane 1: LNCAP, Lane 2: Hela, Lane 3: Jurkat, Lane 4: NIH/3T3