Dynamin 1 Antibody [N9J18]

카탈로그 번호 F2580

인쇄

생물학적 설명

특이성

Dynamin 1 Antibody [N9J18]는 Dynamin 1 단백질의 총 내인성 수준을 검출합니다.
배경

Dynamin 1은 소포 형성에 관여하는 뉴런 엔도사이토시스 단백질이며, Dynamin 2 및 3과 함께 포유류에서 발견되는 세 가지 다이나민 동형 중 하나입니다. Dynamin 1은 주로 뇌에서 발현되는 반면, Dynamin 2는 전반적으로 존재하며, Dynamin 3은 뇌, 폐 및 고환에 국한되어 있습니다. 세 가지 다이나민 모두 N-말단 GTPase 도메인, 번들 신호 요소, 줄기 도메인, 인지질과 결합하는 플렉스트린 상동성 도메인, 그리고 프롤린과 아르기닌 잔기가 풍부한 C-말단 영역으로 구성된 유사한 구조를 공유합니다. 이 단백질들은 막 분열을 촉진하여 엔도사이토시스에서 중요한 역할을 하며 세포골격 조절에도 기여합니다. 다이나민은 액틴과 상호작용하여 라멜리포디아, 등쪽 막 러플, 침입족, 포도좀, 성장 원뿔, 식세포 컵과 같은 구조에서 그 역학에 영향을 미칩니다. Dynamin 1은 미세소관에 특이적으로 결합하여 GTPase 활성을 향상시킵니다. Dynamin 1-3은 별개의 유전자에서 생성되지만, 그 도메인과 기능은 상당히 유사합니다. 족세포에서 Dynamin 1은 Dynamin 2와 유사한 역할을 수행하며, 사구체 여과 장벽의 구조적 무결성에 필수적입니다. Dynamin 1과 2의 족세포 특이적 이중 녹아웃은 심각한 단백뇨와 신부전을 유발합니다. Dynamin은 족세포 발돌기 표면에서 네프린의 엔도사이토시스 및 액틴 필라멘트와의 직접적 및 간접적 상호작용을 통해 발돌기의 구조를 유지하는 데 관여합니다. 또한, Bis-T-23을 사용한 다이나민 올리고머화 증가는 족세포에서 스트레스 섬유 및 초점 부착 형성을 증가시켜 다양한 동물 모델에서 단백뇨 감소를 가져오는 것으로 나타났습니다. 

사용 정보

응용 WB, IHC 희석
WB IHC
1:2000 1:50
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 97 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 1:2000), 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
1242. 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. (Exposure time of at least 60s is recommended)
IHC
Experimental Protocol：
 
Deparaffinization/Rehydration
1. Deparaffinize/hydrate sections:
2. Incubate sections in three washes of xylene for 5 min each.
3. Incubate sections in two washes of 100% ethanol for 10 min each.
4. Incubate sections in two washes of 95% ethanol for 10 min each.
5. Wash sections two times in dH2O for 5 min each.
6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
 
Staining
1. Wash sections in dH2O three times for 5 min each.
2. Incubate sections in 3% hydrogen peroxide for 10 min.
3. Wash sections in dH2O two times for 5 min each.
4. Wash sections in wash buffer for 5 min.
5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
9. Wash sections three times with wash buffer for 5 min each.
10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
12. Immerse slides in dH2O.
13. If desired, counterstain sections with hematoxylin.
14. Wash sections in dH2O two times for 5 min each.
15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
16. Mount sections with coverslips and mounting medium.
 

참조

  • https://pubmed.ncbi.nlm.nih.gov/33070431/

적용 데이터

WB

Selleck 검증

  • F2580-wb
    Lane 1: C6
    Lane 2: NIH/3T3
    Lane 3: SH-SY5Y