|
인용 방법 1. 본문 인용 (재료 및 방법): 2. 주요 자원 표: |
||
|
무료 전화: (877) 796-6397 -- 미국 및 캐나다 전용 -- |
팩스: +1-832-582-8590 주문: +1-832-582-8158 |
기술 지원: +1-832-582-8158 Ext:3 이메일에 주문 번호를 기재해 주십시오. 모든 이메일 문의는 영업일 기준 1일 이내에 답변해 드리기 위해 노력하고 있습니다. |
생물학적 설명
| 특이성 | UCP1 Antibody [L8P13]는 총 UCP1 단백질의 내인성 수준을 인식합니다. 이 항체는 UCP2 또는 UCP3 단백질과 교차 반응하지 않습니다. |
|---|---|
| 배경 | 탈공액 단백질 1(UCP1)은 서모제닌으로도 알려져 있으며, 주로 갈색 지방 조직(BAT)과 베이지색 지방에서 발현되는 33kDa 막관통 단백질입니다. 이곳에서 UCP1은 비떨림 열 발생과 에너지 조절을 촉진합니다. 구조적으로 UCP1은 내부 미토콘드리아 막 내에 5~6개의 막관통 나선 구조를 포함하고 있으며, 장쇄 지방산 결합을 위한 소수성 포켓을 특징으로 하여 양성자 수송을 가능하게 합니다. 기능적으로 UCP1은 산화적 인산화를 탈공액화하여 에너지를 열로 발산하고, 미토콘드리아 막 전위를 조절하며, 활성산소종(ROS)을 감소시키고, 대사 항상성에 기여합니다. UCP1의 발현은 저온 노출, 아드레날린 신호 전달, 노르에피네프린 및 갑상선 호르몬과 같은 호르몬에 의해 조절되며, GDP와 같은 뉴클레오타이드에 의해 억제됩니다. UCP1은 에너지 소비를 증가시키고 ROS 생성을 감소시켜 비만 및 대사 장애와 싸우는 데 중요한 역할을 합니다. |
사용 정보
| 응용 | WB, IHC, IF | 희석 |
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| 반응성 | Mouse, Rat | ||||||||
| 출처 | Rabbit Monoclonal Antibody | MW | 30 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, 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
1211. 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.
|
| 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
|
적용 데이터
IF
Selleck 검증
-
Immunofluorescent analysis of mouse brown adipose tissue tissue using F0765 (green, 1:200) and Hoechst (blue).
IHC
Selleck 검증
-
Immunohistochemical analysis of formalin fixed paraffin embedded mouse fat tissue with F0765 at 1:50 dilution.
WB
Selleck 검증
-
Lane 1: Mouse brown adipose
Lane 2: Mouse liver