데이터시트

생물학적 설명

특이성	CARD11 Antibody [D9F10]는 총 CARD11 단백질의 내인성 수준을 검출합니다.
배경	CARD11 (Caspase Recruitment Domain-containing protein 11)은 B 세포 수용체 (BCR) 및 T 세포 수용체 (TCR)에 의해 활성화되는 신호 전달 경로에 관여하는 핵심 스캐폴드 단백질입니다. BCL10 (B 세포 림프종/백혈병 10) 및 MALT1 (점막 관련 림프 조직 림프종 전위 단백질 1)을 포함하는 CBM 복합체 형성에 도움을 주므로 NF-κB 신호 전달 및 T 세포 활성화에 필수적입니다. 이 복합체는 B 및 T 세포에서 면역 반응 및 세포 생존을 유발하는 신호를 전달하는 데 중요합니다. 단백질 키나아제 C (PKC)에 의한 CARD11의 인산화는 활성화 및 기능에 결정적입니다. 이 인산화는 CARD11이 NF-κB 및 AP-1과 같은 하류 경로를 활성화하는 신호 전달 파트너를 모집할 수 있도록 합니다. CARD11의 돌연변이는 면역 질환을 유발할 수 있으며, 기능 상실 (LOF) 돌연변이는 일반 가변 면역 결핍 (CVID)과 같은 질환과 관련이 있고, 기능 획득 (GOF) 돌연변이는 BENTA 및 미만성 거대 B 세포 림프종 (DLBCL) 및 말초 T 세포 림프종 (PTCL)을 포함한 특정 림프종과 같은 질환과 관련이 있습니다. GOF 돌연변이는 또한 비정상적인 T 세포 활성화 및 면역 체크포인트 기능 장애를 유발하여 면역 조절 장애 및 림프종 발달을 초래합니다.

특이성

CARD11 Antibody [D9F10]는 총 CARD11 단백질의 내인성 수준을 검출합니다.

배경

CARD11 (Caspase Recruitment Domain-containing protein 11)은 B 세포 수용체 (BCR) 및 T 세포 수용체 (TCR)에 의해 활성화되는 신호 전달 경로에 관여하는 핵심 스캐폴드 단백질입니다. BCL10 (B 세포 림프종/백혈병 10) 및 MALT1 (점막 관련 림프 조직 림프종 전위 단백질 1)을 포함하는 CBM 복합체 형성에 도움을 주므로 NF-κB 신호 전달 및 T 세포 활성화에 필수적입니다. 이 복합체는 B 및 T 세포에서 면역 반응 및 세포 생존을 유발하는 신호를 전달하는 데 중요합니다. 단백질 키나아제 C (PKC)에 의한 CARD11의 인산화는 활성화 및 기능에 결정적입니다. 이 인산화는 CARD11이 NF-κB 및 AP-1과 같은 하류 경로를 활성화하는 신호 전달 파트너를 모집할 수 있도록 합니다. CARD11의 돌연변이는 면역 질환을 유발할 수 있으며, 기능 상실 (LOF) 돌연변이는 일반 가변 면역 결핍 (CVID)과 같은 질환과 관련이 있고, 기능 획득 (GOF) 돌연변이는 BENTA 및 미만성 거대 B 세포 림프종 (DLBCL) 및 말초 T 세포 림프종 (PTCL)을 포함한 특정 림프종과 같은 질환과 관련이 있습니다. GOF 돌연변이는 또한 비정상적인 T 세포 활성화 및 면역 체크포인트 기능 장애를 유발하여 면역 조절 장애 및 림프종 발달을 초래합니다.

사용 정보

응용

WB

희석

WB

1:1000

반응성

Human, Mouse

출처

Rabbit Monoclonal Antibody

MW

130 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 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/NP-40 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/NP-40 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: 5%. 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 902. 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 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/NP-40 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/NP-40 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: 5%. 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

902. 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/36960072/

적용 데이터

WB

Selleck 검증

Lane 1: K562
Lane 2: SR
Lane 3: NK92