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Descripción biológica

Especificidad	Mitofusin 1 Antibody [D5A17] detecta niveles endógenos de la proteína Mitofusin-1 total.
Antecedentes	La Mitofusin 1 (MFN1) es un miembro clave de la familia de las GTPasas relacionadas con la dinamina, ubicada en la membrana mitocondrial externa, donde es esencial para la fusión mitocondrial, un proceso vital para mantener la integridad de la red mitocondrial, la homeostasis energética y la supervivencia celular. MFN1 contiene un dominio GTPasa N-terminal que permite la unión e hidrólisis de GTP, dos regiones de repetición de heptadas (HR1 y HR2) que forman estructuras en espiral que median la dimerización y el anclaje entre mitocondrias adyacentes, y dos hélices transmembrana que anclan la proteína dentro de la membrana externa. La HR2 extendida facilita la interacción directa con MFN1 o MFN2 en las mitocondrias vecinas para impulsar una fusión eficiente. MFN1 trabaja en estrecha colaboración con MFN2 y OPA1 para preservar la morfología mitocondrial, estabilizar el ADN mitocondrial (ADNmt), regular la producción de ATP, apoyar las vías de apoptosis y permitir la adaptación metabólica bajo estrés. También promueve la mitofagia, previniendo la acumulación de mitocondrias disfuncionales, y es importante para la embriogénesis y el desarrollo neuronal. La pérdida o disfunción de MFN1 altera la dinámica mitocondrial, causando déficits energéticos, neurodegeneración y enfermedades metabólicas. Aunque las mutaciones de MFN1 son menos comunes que las de MFN2, la deficiencia exacerba la neuropatía tipo Charcot-Marie-Tooth tipo 2A, la atrofia muscular y las condiciones neuroinflamatorias; se observa una expresión alterada de MFN1 en enfermedades como Parkinson, Alzheimer y algunos cánceres.

Especificidad

Mitofusin 1 Antibody [D5A17] detecta niveles endógenos de la proteína Mitofusin-1 total.

Antecedentes

La Mitofusin 1 (MFN1) es un miembro clave de la familia de las GTPasas relacionadas con la dinamina, ubicada en la membrana mitocondrial externa, donde es esencial para la fusión mitocondrial, un proceso vital para mantener la integridad de la red mitocondrial, la homeostasis energética y la supervivencia celular. MFN1 contiene un dominio GTPasa N-terminal que permite la unión e hidrólisis de GTP, dos regiones de repetición de heptadas (HR1 y HR2) que forman estructuras en espiral que median la dimerización y el anclaje entre mitocondrias adyacentes, y dos hélices transmembrana que anclan la proteína dentro de la membrana externa. La HR2 extendida facilita la interacción directa con MFN1 o MFN2 en las mitocondrias vecinas para impulsar una fusión eficiente. MFN1 trabaja en estrecha colaboración con MFN2 y OPA1 para preservar la morfología mitocondrial, estabilizar el ADN mitocondrial (ADNmt), regular la producción de ATP, apoyar las vías de apoptosis y permitir la adaptación metabólica bajo estrés. También promueve la mitofagia, previniendo la acumulación de mitocondrias disfuncionales, y es importante para la embriogénesis y el desarrollo neuronal. La pérdida o disfunción de MFN1 altera la dinámica mitocondrial, causando déficits energéticos, neurodegeneración y enfermedades metabólicas. Aunque las mutaciones de MFN1 son menos comunes que las de MFN2, la deficiencia exacerba la neuropatía tipo Charcot-Marie-Tooth tipo 2A, la atrofia muscular y las condiciones neuroinflamatorias; se observa una expresión alterada de MFN1 en enfermedades como Parkinson, Alzheimer y algunos cánceres.

Información de uso

Aplicación

WB, IP

Dilución

WB	IP
1:1000	1:30

Reactividad

Mouse, Rat

Fuente

Rabbit Monoclonal Antibody

MW

84 kDa

Tampón de almacenamiento

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Almacenamiento
(Desde la fecha de recepción)

-20°C (avoid freeze-thaw cycles), 2 years

Métodos experimentales
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, 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 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.

Métodos experimentales

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, 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

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.

Referencias

https://pubmed.ncbi.nlm.nih.gov/30647902/
https://pubmed.ncbi.nlm.nih.gov/27920125/

Datos de aplicación

WB

Validado por Selleck

Lane 1: NIH/3T3, Lane 2: Neuro-2a, Lane 3: Mouse brain, Lane 4: Rat brain