QKI Antibody [M12N24]

카탈로그 번호 F2558

인쇄

생물학적 설명

특이성

QKI Antibody [M12N24]는 QKI 단백질의 총 내인성 수준을 검출합니다.
배경

RNA 결합 단백질(RBP)은 전령 RNA(pre-mRNA)의 전사 후 처리, 세포 내 운반 및 mRNA 안정성 조절을 포함한 다양한 생물학적 및 생리적 과정에서 필수적인 역할을 합니다. 주목할 만한 RBP 중 하나는 QKI로, Quaking이라고도 알려져 있으며, 신호 전달 및 RNA 활성화(STAR) 계열의 일원이자 이질 핵 리보핵단백질 K(hnRNP K) 상동성 도메인 단백질 계열의 일부입니다. QKI는 QKI-5, QKI-6, QKI-7의 세 가지 주요 대체 스플라이싱 이성질체로 존재하며, 각각 카복시 말단 도메인이 다릅니다. 모든 이성질체는 공통된 RNA 결합 능력을 공유하지만, pre-mRNA 스플라이싱, mRNA 안정성 또는 운반을 조절하는 특정 역할은 세포 유형에 따라 다릅니다. QKI 유전자는 인간 염색체 6번과 마우스 염색체 17번에 위치합니다. 이 유전자는 KH 도메인 내에 RNA 결합 모티프를 포함하고 있으며, 두 개의 QUA 도메인(QUA1 및 QUA2)으로 둘러싸인 단백질을 암호화합니다. QKI는 신경학적 질환 및 심혈관 발달, 단핵구-대식세포 분화, 뼈 대사 및 암 진행을 포함한 더 넓은 생물학적 시스템에서 중요한 역할을 하는 것으로 알려져 있습니다. 각 QKI 이성질체는 뚜렷한 생물학적 기능을 나타냅니다. QKI-5는 핵 위치 신호를 포함하며 주로 pre-mRNA 스플라이싱 조절에 관여합니다. 이와 대조적으로 QKI-6과 QKI-7은 핵 위치 신호가 없으며 안정성 및 운반과 같은 mRNA 전사 후 과정 조절에 더 적극적으로 관여합니다. 이러한 이성질체는 특정 맥락에서 다양한 생물학적 기능에 기여합니다. QKI는 널리 발현되지만, 배아 발달 중 심장 및 중추 신경계에 특히 풍부하게 존재하며, 여기서 근원섬유 형성 및 심장 기능 보호에 핵심적인 역할을 합니다. 혈관 평활근 세포(VSMC)에서 QKI 발현은 혈관 손상에 대한 반응으로 강력하게 유도됩니다. 이는 평활근 분화의 주요 전사 인자인 Myocd의 대체 스플라이싱을 조절함으로써 VSMC 탈분화에 기여합니다. 이는 발달, 손상 반응 및 질병 진행에서 QKI의 다면적인 역할을 강조합니다. 

사용 정보

응용 WB, IP, IF 희석
WB IP IF
1:1000 -1: 10000 1:50 - 1:100 1:50 - 1:100
반응성 Human, Mouse
출처 Rabbit Monoclonal Antibody MW 37 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
1240. 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 90s is recommended)
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.

참조

  • https://pubmed.ncbi.nlm.nih.gov/34222237/

적용 데이터

WB

Selleck 검증

  • F2558-wb
    Lane 1: Neuro 2a
    Lane 2: HeLa
    Lane 3: K562

IF

Selleck 검증

  • F2558-IF
    Immunofluorescent analysis of Neuro-2a cells using F2558 (green, 1:50), Hoechst (blue) and tubulin (Red).