ENO1 Antibody [N16N23]

카탈로그 번호 F2363

인쇄

생물학적 설명

특이성

ENO1 Antibody [N16N23]는 총 ENO1 단백질의 내인성 수준을 인식합니다.
배경

엔올라제 1(ENO1)은 해당 경로에서 중요한 역할을 하는 다기능 효소로, 2-포스포글리세레이트가 포스포엔올피루브산으로 전환되는 것을 촉진하여 ATP 생성의 핵심 단계를 수행합니다. 구조적으로 ENO1은 활성 부위를 둘러싸는 L1, L2, L3 루프를 가진 폐쇄형 구조를 취하며, 이 활성 부위에는 효소 기능에 필수적인 마그네슘 이온이 포함되어 있습니다. ENO1은 또한 플라스미노겐에 결합하여 플라스민으로 전환시키고, 이는 기질 분해 효소를 활성화하며 조직 리모델링, 세포 침입 및 전이에서 역할을 합니다. ENO1은 핵 DNA 결합 단백질로 기능하며 유전자 전사를 조절하고 자가면역 질환에서 자가항원으로 작용합니다. ENO1의 표면 특징은 DNA 결합 능력과 플라스미노겐 결합에 기여하는 양전하를 띠는 잔기를 가지고 있습니다. 이는 세포 성장, 분화, 이동, 세포자멸사 및 세포골격 조절과 같은 다양한 세포 기능에 관여하며, 이는 조직 발달, 상처 치유 및 면역 반응에 중요합니다. ENO1은 유방암, 폐암, 간암과 같은 암에서 종종 과발현되어 종양 진행 및 전이를 촉진합니다. 또한 자가면역 질환에서도 역할을 하며, 류마티스 관절염 및 루푸스와 같은 질환에서 항-ENO1 항체가 검출됩니다. 기생충 감염에서는 ENO1이 플라스모디움 팔시파룸 메로조이트의 표면에 존재하여 면역 인식 및 기생충 생존에 영향을 미칩니다. 또한 ENO1은 정자 운동성 및 ATP 생산에 기여하여 남성 생식 능력에 필수적이며, 근육 발달 및 재생에 관여합니다.

사용 정보

응용 WB, IP, IF, FCM 희석
WB IP IF FCM
1:1000-1:10000 1:50 1:60 1:50
반응성 Human, Mouse, Rat
출처 Rabbit Monoclonal Antibody MW 47 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
1225. 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.
 
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/18560153/
  • https://pubmed.ncbi.nlm.nih.gov/29767008/

적용 데이터

WB

Selleck 검증

  • F2363-wb
    Lane 1: MCF7
    Lane 2: Jurkat
    Lane 3: A431
    Lane 4: HeLa
    Lane 5: C2C12
    Lane 6: NIH/3T3

IF

Selleck 검증

  • F2363-IF
    Immunofluorescent analysis of MCF-7 cells using F2363 (green, 1:50), Hoechst (blue) and tubulin (Red).