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생물학적 설명

특이성	Caspase-1 Antibody [C2H20]는 총 Caspase-1 단백질의 내인성 수준을 검출합니다.
배경	Caspase-1 (인터루킨-1β 전환 효소, ICE)은 Caspase-4, -5, -11, -12와 함께 Caspase 계열의 염증성 시스테인 프로테아제로 분류되며, 선천성 면역 반응에 필수적인 비활성 45 kDa 프로엔자임으로 합성됩니다. 이 단백질은 인플라마좀 조립을 위한 N-말단 Caspase 모집 도메인(CARD), 촉매 시스테인 잔기를 포함하는 큰 p20 소단위체, 그리고 작은 p10 소단위체를 특징으로 하며, 이들이 근접 유도 이량화 및 자가 단백질 분해를 통해 활성 이종사량체 (p20)₂(p10)₂를 형성합니다. Caspase-1은 병원체 관련 분자 패턴 또는 손상 신호에 의해 유발된 다중 단백질 인플라마좀(NLRP3, AIM2, NLRC4/ASC) 내에서 활성화되어, pro-IL-1β 및 pro-IL-18을 발열, 염증 및 적응 면역을 유도하는 성숙한 전염증성 사이토카인으로 특이적으로 절단하고, 가스더민 D를 처리하여 N-말단 구멍 형성을 통해 파이롭토시스를 실행하여 용해성 세포 사멸 및 IL-1 방출을 유발합니다. Caspase-1 결핍은 사이토카인 성숙을 선택적으로 손상시키고 Apoptosis에 영향을 미치지 않으면서 내독소 쇼크에 대한 저항성을 부여합니다.

특이성

Caspase-1 Antibody [C2H20]는 총 Caspase-1 단백질의 내인성 수준을 검출합니다.

배경

Caspase-1 (인터루킨-1β 전환 효소, ICE)은 Caspase-4, -5, -11, -12와 함께 Caspase 계열의 염증성 시스테인 프로테아제로 분류되며, 선천성 면역 반응에 필수적인 비활성 45 kDa 프로엔자임으로 합성됩니다. 이 단백질은 인플라마좀 조립을 위한 N-말단 Caspase 모집 도메인(CARD), 촉매 시스테인 잔기를 포함하는 큰 p20 소단위체, 그리고 작은 p10 소단위체를 특징으로 하며, 이들이 근접 유도 이량화 및 자가 단백질 분해를 통해 활성 이종사량체 (p20)₂(p10)₂를 형성합니다. Caspase-1은 병원체 관련 분자 패턴 또는 손상 신호에 의해 유발된 다중 단백질 인플라마좀(NLRP3, AIM2, NLRC4/ASC) 내에서 활성화되어, pro-IL-1β 및 pro-IL-18을 발열, 염증 및 적응 면역을 유도하는 성숙한 전염증성 사이토카인으로 특이적으로 절단하고, 가스더민 D를 처리하여 N-말단 구멍 형성을 통해 파이롭토시스를 실행하여 용해성 세포 사멸 및 IL-1 방출을 유발합니다. Caspase-1 결핍은 사이토카인 성숙을 선택적으로 손상시키고 Apoptosis에 영향을 미치지 않으면서 내독소 쇼크에 대한 저항성을 부여합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:200

반응성

Mouse

출처

Rabbit Monoclonal Antibody

MW

48 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. ( 0.45 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 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. ( 0.45 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

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/33116753/
https://pubmed.ncbi.nlm.nih.gov/19221555/

적용 데이터

WB

Selleck 검증

Lane 1: A20, Lane 2: EL4, Lane 3: J774A.1