데이터시트

생물학적 설명

특이성	Cox1 Antibody [N11K4]는 총 Cox1 단백질의 내인성 수준을 검출합니다.
배경	사이클로옥시게나제-1(COX-1)은 프로스타글란딘 G/H 합성효소 1(PTGS1)로도 알려져 있으며, 프로스타노이드 생합성의 핵심 단계인 아라키돈산의 프로스타글란딘 H2로의 전환을 촉매하는 사이클로옥시게나제 계열에 속하는 막 결합형, 헴 의존성 효소입니다. COX-1은 각 서브유닛당 세 가지 주요 도메인으로 구성된 호모다이머입니다: 표피 성장 인자 도메인, 막 결합 도메인, 그리고 사이클로옥시게나제와 퍼옥시다제 활성 부위를 모두 포함하는 촉매 도메인으로, 헴을 보조 인자로 사용하여 지질 이중층의 단일 리플렛에 통합됩니다. 이 효소의 사이클로옥시게나제 활성은 아라키돈산을 산소화하여 PGG2를 형성하고, 이는 후속적으로 퍼옥시다제 기능에 의해 PGH2로 환원되어 염증, 혈소판 응집 및 혈관 항상성 조절에 필수적인 분자인 프로스타글란딘과 트롬복산의 합성을 시작합니다. COX-1은 대부분의 조직에서 구성적으로 발현되며, 정상 생리적 조건하에서 위 점막의 무결성, 신장 혈류 및 혈소판 기능 유지에 중요한 역할을 합니다. 그 활성은 지질 과산화물 수준에 의해 조절되며, 낮은 아라키돈산 농도에서 독특한 음성 알로스테릭 조절을 나타내어 프로스타노이드 합성을 미세 조정할 수 있습니다. COX-1은 또한 아스피린과 같은 비스테로이드성 항염증제(NSAID)에 의해 비가역적으로 억제되는데, 아스피린은 활성 부위 근처의 세린 잔기를 변형시켜 트롬복산 A2 형성을 방지하고 혈소판 응집 및 염증을 감소시킵니다.

특이성

Cox1 Antibody [N11K4]는 총 Cox1 단백질의 내인성 수준을 검출합니다.

배경

사이클로옥시게나제-1(COX-1)은 프로스타글란딘 G/H 합성효소 1(PTGS1)로도 알려져 있으며, 프로스타노이드 생합성의 핵심 단계인 아라키돈산의 프로스타글란딘 H2로의 전환을 촉매하는 사이클로옥시게나제 계열에 속하는 막 결합형, 헴 의존성 효소입니다. COX-1은 각 서브유닛당 세 가지 주요 도메인으로 구성된 호모다이머입니다: 표피 성장 인자 도메인, 막 결합 도메인, 그리고 사이클로옥시게나제와 퍼옥시다제 활성 부위를 모두 포함하는 촉매 도메인으로, 헴을 보조 인자로 사용하여 지질 이중층의 단일 리플렛에 통합됩니다. 이 효소의 사이클로옥시게나제 활성은 아라키돈산을 산소화하여 PGG2를 형성하고, 이는 후속적으로 퍼옥시다제 기능에 의해 PGH2로 환원되어 염증, 혈소판 응집 및 혈관 항상성 조절에 필수적인 분자인 프로스타글란딘과 트롬복산의 합성을 시작합니다. COX-1은 대부분의 조직에서 구성적으로 발현되며, 정상 생리적 조건하에서 위 점막의 무결성, 신장 혈류 및 혈소판 기능 유지에 중요한 역할을 합니다. 그 활성은 지질 과산화물 수준에 의해 조절되며, 낮은 아라키돈산 농도에서 독특한 음성 알로스테릭 조절을 나타내어 프로스타노이드 합성을 미세 조정할 수 있습니다. COX-1은 또한 아스피린과 같은 비스테로이드성 항염증제(NSAID)에 의해 비가역적으로 억제되는데, 아스피린은 활성 부위 근처의 세린 잔기를 변형시켜 트롬복산 A2 형성을 방지하고 혈소판 응집 및 염증을 감소시킵니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse

출처

Rabbit Monoclonal Antibody

MW

70 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/12432914/
https://pubmed.ncbi.nlm.nih.gov/11597987/

적용 데이터

WB

Selleck 검증

Lane 1: C2C12, Lane 2: Hela