QKI Antibody [M12N24]

Katalog-Nr. F2558

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Biologische Beschreibung

Spezifität

QKI Antibody [M12N24] weist die gesamten endogenen Mengen an QKI-Protein nach.
Hintergrund

RNA-bindende Proteine (RBPs) spielen eine wesentliche Rolle in verschiedenen biologischen und physiologischen Prozessen, einschließlich der Regulation der posttranskriptionellen Verarbeitung von prä-mRNA, des intrazellulären Transports und der mRNA-Stabilität. Ein bemerkenswertes RBP ist QKI, auch bekannt als Quaking, ein Mitglied der STAR-Familie (Signal Transduction and Activation of RNA), die auch zur Proteinfamilie der heterogenen nukleären Ribonukleoprotein K (hnRNP K)-Homologiedomäne gehört. QKI existiert in drei Haupt-alternativ gespleißten Isoformen – QKI-5, QKI-6 und QKI-7 – die sich jeweils in ihren Carboxy-terminalen Domänen unterscheiden. Während alle Isoformen eine gemeinsame RNA-Bindungsfähigkeit teilen, variieren ihre spezifischen Rollen bei der Regulierung des prä-mRNA-Spleißens, der mRNA-Stabilität oder des Transports zelltypspezifisch. Das QKI-Gen befindet sich auf dem menschlichen Chromosom 6 und dem Maus-Chromosom 17. Es kodiert Proteine, die ein RNA-bindendes Motiv innerhalb der KH-Domäne enthalten, flankiert von zwei QUA-Domänen (QUA1 und QUA2). QKI ist bekannt für seine kritische Rolle bei neurologischen Erkrankungen und breiteren biologischen Systemen, einschließlich der kardiovaskulären Entwicklung, der Monozyten-zu-Makrophagen-Differenzierung, des Knochenstoffwechsels und der Krebsentwicklung. Jede QKI-Isoform weist unterschiedliche biologische Funktionen auf. QKI-5 enthält ein nukleäres Lokalisierungssignal und ist überwiegend an der Regulierung des prä-mRNA-Spleißens beteiligt. Im Gegensatz dazu fehlen QKI-6 und QKI-7 nukleäre Lokalisierungssignale und sind aktiver an der Regulierung posttranskriptioneller mRNA-Prozesse wie Stabilität und Transport beteiligt. Diese Isoformen tragen in spezifischen Kontexten zu verschiedenen biologischen Funktionen bei. QKI ist weit verbreitet, ist aber während der Embryonalentwicklung besonders im Herzen und im zentralen Nervensystem angereichert, wo es eine Schlüsselrolle beim Schutz der Myofibrillogenese und der Herzfunktion spielt. In vaskulären glatten Muskelzellen (VSMCs) wird die QKI-Expression als Reaktion auf Gefäßverletzungen stark induziert. Es trägt zur VSMC-Dedifferenzierung bei, indem es das alternative Spleißen von Myocd reguliert, einem Master-Transkriptionsfaktor bei der Differenzierung glatter Muskeln. Dies unterstreicht die vielfältigen Rollen von QKI bei Entwicklung, Verletzungsreaktion und Krankheitsentwicklung. 

Nutzungsinformationen

Anwendung WB, IP, IF Verdünnung
WB IP IF
1:1000 -1: 10000 1:50 - 1:100 1:50 - 1:100
Reaktivität Human, Mouse
Quelle Rabbit Monoclonal Antibody MW 37 kDa
Lagerpuffer PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃
Lagerung
(Ab dem Datum des Erhalts)		 -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.

Referenzen

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

Anwendungsdaten

WB

Validiert von Selleck

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

IF

Validiert von Selleck

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