데이터시트

생물학적 설명

특이성	Arf6 Antibody [N23E24]는 총 Arf6 단백질의 내인성 수준을 검출합니다.
배경	광범위한 Ras 초과족에 속하는 ADP-리보실화 인자(Arf) 단백질 계열은 작은 GTPase를 포함합니다. 인간에서 이 계열은 Arf-유사 단백질(Arls), Sar 단백질 및 6가지 Arf(Arf1-Arf6)를 포함합니다. 이 Arf 단백질은 서열 상동성에 따라 세 가지 클래스로 더 분류됩니다: 클래스 I (Arf1-3), 클래스 II (Arf4-5), 클래스 III (Arf6). Arf6는 세포막, 세포질 및 엔도솜 막에 위치하며 N-말단 미리스틸화를 거칩니다. Arf6는 세포 내 소포 수송에서 중요한 역할을 하며, 클라트린 의존적 또는 비의존적 엔도사이토시스를 통해 내부화된 다양한 화물 유형의 재활용을 조절합니다. 또한 Arf6는 PIP 5-키나제 및 포스포리파제 D와 같은 지질 변형 효소를 활성화하고, 액틴 중합을 촉진하며, 다양한 세포 과정을 조절합니다. Arf6는 병원체 식세포 작용에 중요한 역할을 하며, 식세포 작용을 방해하거나 세포 내 병원체 흡수를 유도하기 위해 병원체에 의해 직접 또는 간접적으로 표적이 될 수 있습니다. 또한 Arf6는 Toll-유사 수용체 신호 전달 및 NADPH 산화효소 활성화에 관여합니다.

특이성

Arf6 Antibody [N23E24]는 총 Arf6 단백질의 내인성 수준을 검출합니다.

배경

광범위한 Ras 초과족에 속하는 ADP-리보실화 인자(Arf) 단백질 계열은 작은 GTPase를 포함합니다. 인간에서 이 계열은 Arf-유사 단백질(Arls), Sar 단백질 및 6가지 Arf(Arf1-Arf6)를 포함합니다. 이 Arf 단백질은 서열 상동성에 따라 세 가지 클래스로 더 분류됩니다: 클래스 I (Arf1-3), 클래스 II (Arf4-5), 클래스 III (Arf6). Arf6는 세포막, 세포질 및 엔도솜 막에 위치하며 N-말단 미리스틸화를 거칩니다. Arf6는 세포 내 소포 수송에서 중요한 역할을 하며, 클라트린 의존적 또는 비의존적 엔도사이토시스를 통해 내부화된 다양한 화물 유형의 재활용을 조절합니다. 또한 Arf6는 PIP 5-키나제 및 포스포리파제 D와 같은 지질 변형 효소를 활성화하고, 액틴 중합을 촉진하며, 다양한 세포 과정을 조절합니다. Arf6는 병원체 식세포 작용에 중요한 역할을 하며, 식세포 작용을 방해하거나 세포 내 병원체 흡수를 유도하기 위해 병원체에 의해 직접 또는 간접적으로 표적이 될 수 있습니다. 또한 Arf6는 Toll-유사 수용체 신호 전달 및 NADPH 산화효소 활성화에 관여합니다.

사용 정보

응용

WB, IP

희석

WB	IP
1:1000	1:50

반응성

Human, Mouse, Rat, Monkey

출처

Rabbit 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 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. ( 0.22 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 1:1000), 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 588. 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. ( 0.22 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 1:1000), 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

588. 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/31060328/

적용 데이터

WB

Selleck 검증

Lane 1: NIH3T3
Lane 2: 293
Lane 3: C6
Lane 4: Raw264.7
Lane 5: COS-7