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인용 방법 1. 본문 인용 (재료 및 방법): 2. 주요 자원 표: |
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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 | Heme Oxygenase 1 Antibody [G7A14]는 총 Heme Oxygenase 1 단백질의 내인성 수준을 감지합니다. 이 항체는 파라핀 절편의 면역형광에는 권장되지 않습니다. |
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| 배경 | 헤모옥시게나제-1(HO-1)은 스트레스 반응 단백질이자 세포 및 조직 항상성을 유지하는 데 중요한 역할을 하는 대사 효소입니다. 또한 면역 반응, 숙주 방어 메커니즘 및 염증 해결의 핵심 조절자 역할을 합니다. 헤모옥시게나제에는 유도성 HO-1과 구성적으로 발현되는 HO-2의 두 가지 주요 동형이 있습니다. 둘 다 헴을 빌리베르딘-IXα(BV), 2가 철(Fe²⁺) 및 일산화탄소(CO)의 세 가지 부산물로 분해하는 것을 촉매합니다. 빌리베르딘은 빌리베르딘 환원효소(BVR)에 의해 빌리루빈-IXα(BR)로 환원됩니다. HO-1은 고도로 유도 가능하며 그 발현은 산화 스트레스, 헴 축적 및 다양한 식물성 항산화제를 포함한 광범위한 자극에 의해 유발됩니다. 이러한 유도는 주로 전사 인자 Nrf2(NF-E2 관련 인자-2)에 의해 조절되며 헴 의존적 방식으로 Bach-1에 의해 억제됩니다. 또한 HMOX1 프로모터 영역의 유전적 변이는 여러 인체 질병에 대한 감수성과 관련이 있습니다. HO-1은 지질 과산화를 촉매할 수 있는 친산화 분자인 헴을 분해하여 염증 환경에서 보호 기능을 수행하여 산화 손상을 줄입니다. 이 과정에서 방출된 철은 과도한 철을 격리하는 페리틴 합성을 촉진하거나 특정 조건에서 페롭토시스에 기여할 수 있습니다. 또한 헴 유래 대사산물인 빌리베르딘과 빌리루빈은 항산화 및 면역 조절 효과를 발휘하여 HO-1의 세포 보호 역할을 더욱 지지합니다. |
사용 정보
| 응용 | WB, IP, IF, FCM | 희석 |
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| 반응성 | Mouse, Rat, Human | ||||||||
| 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 | ||||||
| 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.
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| IF |
Experimental Protocol：
Specimen Preparation
1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
NOTE: Paraformaldehyde is toxic, use only in a fume hood.
2. Fix cells for 15 min at room temperature.
3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
4. Proceed with Immunostaining.
Immunostaining
1. Add theblocking buffer and incubate for 60 min at RT.
2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
3. Aspirate blocking solution, apply diluted primary antibody.
4. Incubate overnight at 4°C.
5. Rinse three times in 1X PBS for 5 min each.
6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
7. Rinse three times in 1X PBS for 5 min each.
8. Mount slides usingmounting medium with DAPI and cover with coverslips.
9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.
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| 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.
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참조
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적용 데이터
WB
Selleck 검증
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Lane 1: HEK-293, Lane 2: NIH/3T3, Lane 3: A549, Lane 4: Rat spleen
IF
Selleck 검증
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Immunofluorescent analysis of HepG2 cells using F3640 (green, 1:100), Hoechst (blue) and tubulin (Red).