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생물학적 설명

특이성	Alix Antibody [A13D20]는 총 Alix 단백질의 내인성 수준을 인식합니다.
배경	Alix는 Pdcd6ip 또는 AIP1(apoptosis-linked gene-2 (ALG-2)-interacting protein 1)으로도 알려져 있으며, 다면적인 구조를 가진 스캐폴드 단백질입니다. 이는 N-말단 Bro1 도메인, 중앙 V자형 도메인 및 C-말단 프롤린이 풍부한 도메인(PRD)의 세 가지 주요 모듈로 구성됩니다. 그러나 C-말단 PRD는 거의 알려져 있지 않고 매우 무질서합니다. PRD는 많은 진핵생물 단백질에서 발견되는 일반적인 모듈입니다. Alix는 주로 두 가지 주요 기능과 관련이 있습니다. 첫째, ESCRT(endosomal sorting complex required for transport) 기계의 구성 요소와 상호 작용하여 ESCRTIII의 모집을 촉진합니다. 이러한 관여는 엔도사이토시스, 막 수용체 재활용, 다중소포체(MVB) 및 엑소좀 형성, 바이러스 출아, 세포 분열 및 세포막 복구와 같은 다양한 세포 과정에 기여합니다. 둘째, Alix는 F-액틴 및 F-액틴 상호 작용 단백질에 결합하여 액틴 세포골격 리모델링에 중요한 역할을 합니다. 그 형태, 결합 파트너와의 상호 작용 및 활성은 유비퀴틴화, 인산화 및 팔미토일화를 포함한 번역 후 변형에 의해 조절됩니다.

특이성

Alix Antibody [A13D20]는 총 Alix 단백질의 내인성 수준을 인식합니다.

배경

Alix는 Pdcd6ip 또는 AIP1(apoptosis-linked gene-2 (ALG-2)-interacting protein 1)으로도 알려져 있으며, 다면적인 구조를 가진 스캐폴드 단백질입니다. 이는 N-말단 Bro1 도메인, 중앙 V자형 도메인 및 C-말단 프롤린이 풍부한 도메인(PRD)의 세 가지 주요 모듈로 구성됩니다. 그러나 C-말단 PRD는 거의 알려져 있지 않고 매우 무질서합니다. PRD는 많은 진핵생물 단백질에서 발견되는 일반적인 모듈입니다. Alix는 주로 두 가지 주요 기능과 관련이 있습니다. 첫째, ESCRT(endosomal sorting complex required for transport) 기계의 구성 요소와 상호 작용하여 ESCRTIII의 모집을 촉진합니다. 이러한 관여는 엔도사이토시스, 막 수용체 재활용, 다중소포체(MVB) 및 엑소좀 형성, 바이러스 출아, 세포 분열 및 세포막 복구와 같은 다양한 세포 과정에 기여합니다. 둘째, Alix는 F-액틴 및 F-액틴 상호 작용 단백질에 결합하여 액틴 세포골격 리모델링에 중요한 역할을 합니다. 그 형태, 결합 파트너와의 상호 작용 및 활성은 유비퀴틴화, 인산화 및 팔미토일화를 포함한 번역 후 변형에 의해 조절됩니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat, Monkey

출처

Mouse Monoclonal Antibody

MW

95 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: 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 24. 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: 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

24. 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/36030822/

적용 데이터

WB

Selleck 검증

Lane 1: Hela
Lane 2: A10
Lane 3: NIH/3T3
Lane 4: COS