MAVS Antibody [P1P22]

카탈로그 번호 F1399

인쇄

생물학적 설명

특이성

MAVS Antibody [P1P22]는 총 MAVS 단백질의 내인성 수준을 인식합니다. 면역조직화학 분석 결과 마우스 뇌에서 비특이적 핵 염색과 마우스 위 상피의 정점막 염색이 관찰되었습니다.
배경

MAVS는 미토콘드리아, 퍼옥시좀, 미토콘드리아 관련 ER 막(MAMs)에서 RIG-I 유사 수용체(RLR) 신호 전달에 관여하는 어댑터 단백질로, RNA 바이러스에 대한 선천 면역에 필수적입니다. MAVS는 RLR 신호 전달에 의해 조절되는 대사 재프로그래밍에 중요합니다. 퍼옥시좀 MAVS는 IRF1을 통해 선택적으로 Type III IFN 발현을 유도하며, 미토콘드리아 MAVS는 Type I IFN 및 ISG 발현을 유도합니다. MAVS는 HK2와 상호 작용하여 비감염 세포에서 글루코스 흐름을 해당 과정으로 보냅니다. 또한 퍼옥시좀에서 G6PD와 상호 작용하여 TRAF6 및 IRF1과 함께 신호전달복합체(signalosome)를 형성하여 글루코스 흐름을 펜토스 인산 경로(PPP) 및 Type III IFN 생성으로 제어합니다. 유비퀴틴화, 응집 및 게라닐게라닐화와 같은 번역 후 변형은 MAVS 기능을 조절합니다. MAVS는 해당 과정에서 PPP 및 헥소사민 생합성 경로(HBP)로의 전환을 유도하며, 퍼옥시좀 MAVS는 PPP로의 글루코스 흐름 및 Type III IFN 발현을 처리하고, MAMs에 위치한 MAVS는 HBP로의 글루코스 흐름 및 Type I IFN 발현을 관리합니다.

사용 정보

응용 WB, IP, IHC, IF 희석
WB IP IHC IF
1:1000 1:100 1:200 - 1:800 1:100 - 1:400
반응성 Mouse
출처 Rabbit Monoclonal Antibody MW 75 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
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
655. 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.
IF
Experimental Protocol:
 
Sample Preparation
1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
 
Fixation
1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
2. Wash the sample with PBS for 3 times, 3 minutes each time.
 
Permeabilization
1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
Wash the sample with PBS for 3 times, 3 minutes each time.
 
Blocking
Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
 
Immunofluorescence Staining (Day 1)
1. Remove the blocking solution and add the diluted primary antibody.
2. Incubate the sample in a humidified chamber at 4°C overnight.
 
Immunofluorescence Staining (Day 2)
1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
5. Wash with PBST for 3 times, 5 minutes each time.
 
Mounting
1. Mount the sample with an anti-fade mounting medium.
2. Allow the slide to dry at room temperature overnight in the dark.
3. Store the slide in a slide storage box at 4°C, protected from light.

참조

  • https://pubmed.ncbi.nlm.nih.gov/37660168/

적용 데이터

WB

Selleck 검증

  • F1399-wb
    Lane 1: Neuro-2a
    Lane 2: BA/F3
    Lane 3: C2C12
    Lane 4: Hepa 1-6
    Lane 5: NIH/3T3

IHC

Selleck 검증

  • F1399-IHC1
    Immunohistochemical analysis of formalin fixed paraffin embedded human Breast cancer tissue with F1399 at 1/200 dilution.

IF

Selleck 검증

  • F1399-IF
    Immunofluorescent analysis of C2C12 cells using F1399 (green, 1:100), Hoechst (blue) and tubulin (Red).