SIRT5 Antibody [G24H6]

카탈로그 번호 F3785

인쇄

생물학적 설명

특이성

SIRT5 Antibody [G24H6]는 총 SIRT5 단백질의 내인성 수준을 검출합니다.
배경

SIRT5는 세포 분화, 세포자멸사, 대사, 노화, 면역 반응, 산화 스트레스 제어 및 미토콘드리아 활동을 포함한 다양한 생리 기능에 관여하는 중요한 조절자입니다. 광범위한 영향으로 인해 SIRT5는 다양한 관련 질병에 대한 유망한 치료 표적으로 부상했습니다. 특히 SIRT5는 암 생물학에서 이중 역할을 합니다. 비소세포폐암(NSCLC)에서 그 발현 패턴은 상황에 따라 다릅니다. 높은 SIRT5 수준은 SUN2 억제와 함께 암세포 증식을 촉진할 수 있습니다. 반대로 NSCLC에서 SIRT5 발현이 감소하면 STAT3 아세틸화가 증가하여 ATP 생성을 촉진하고 종양 성장을 지원합니다. 인간 Sirt5 유전자는 6p3 염색체에 위치하며 약 34 kDa의 분자량을 가진 4가지 단백질 동형인 SIRT5˄iso1, SIRT5˄iso2, SIRT5˄iso3 및 SIRT5˄iso4를 암호화합니다. SIRT5˄iso1–3 동형은 주로 미토콘드리아에 국한되는 반면, SIRT5˄iso4는 주로 세포질에 존재합니다. Sirt5 유전자는 인간 조직 전반에 걸쳐 광범위하게 발현되지만 동형 분포는 다릅니다. SIRT5˄iso1은 지방, 방광, 기관을 제외한 거의 모든 인간 조직에서 발현되며, 뇌와 신장에서 가장 풍부합니다. SIRT5˄iso2는 뇌에서 가장 흔하게 발견되지만 다른 조직에서는 덜합니다. SIRT5˄iso3–4는 주로 뇌와 신장에서 발현됩니다. 구조적으로 SIRT5는 선택적 기질 표적화를 가능하게 하는 독특한 특징을 가지고 있습니다. 그 탈아실화 효소 활성은 펩타이드 사슬에서 아세틸, 숙시닐, 말로닐 및 글루타릴 그룹을 특이적으로 제거하여 다양한 대사 및 조절 경로를 조절합니다.

사용 정보

응용 WB, IP, IHC, FCM 희석
WB IP IHC FCM
1:1000 1:30 1:100 1:50
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 33 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
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.
 
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, 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
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/39979670/

적용 데이터

WB

Selleck 검증

  • F3785-wb
    Lane 1: HepG2, Lane 2: Huh7, Lane 3: Mouse heart, Lane 4: Rat heart