Fiche technique

Description biologique

Spécificité	Mitofusin 1 Antibody [D5A17] détecte les niveaux endogènes de la protéine Mitofusin-1 totale.
Contexte	La Mitofusin 1 (MFN1) est un membre clé de la famille des GTPases apparentées à la dynamine, située sur la membrane mitochondriale externe, où elle est essentielle à la fusion mitochondriale, un processus vital pour maintenir l'intégrité du réseau mitochondrial, l'homéostasie énergétique et la survie cellulaire. MFN1 contient un domaine GTPase N-terminal permettant la liaison et l'hydrolyse du GTP, deux régions de répétition d'heptapeptides (HR1 et HR2) qui forment des structures enroulées-bobinées médiatisant la dimérisation et l'ancrage entre les mitochondries adjacentes, et deux hélices transmembranaires ancrant la protéine dans la membrane externe. La HR2 étendue facilite l'interaction directe avec MFN1 ou MFN2 sur les mitochondries voisines pour favoriser une fusion efficace. MFN1 travaille en étroite collaboration avec MFN2 et OPA1 pour préserver la morphologie mitochondriale, stabiliser l'ADN mitochondrial (ADNmt), réguler la production d'ATP, soutenir les voies d'apoptose et permettre l'adaptation métabolique en cas de stress. Elle favorise également la mitophagie, empêchant l'accumulation de mitochondries dysfonctionnelles, et est importante pour l'embryogenèse et le développement neuronal. La perte ou le dysfonctionnement de MFN1 altère la dynamique mitochondriale, entraînant des déficits énergétiques, une neurodégénérescence et des maladies métaboliques. Bien que les mutations de MFN1 soient moins fréquentes que celles de MFN2, une déficience exacerbe la neuropathie de type Charcot-Marie-Tooth de type 2A, l'atrophie musculaire et les affections neuroinflammatoires ; une expression altérée de MFN1 est observée dans des maladies telles que Parkinson, Alzheimer et certains cancers.

Spécificité

Mitofusin 1 Antibody [D5A17] détecte les niveaux endogènes de la protéine Mitofusin-1 totale.

Contexte

La Mitofusin 1 (MFN1) est un membre clé de la famille des GTPases apparentées à la dynamine, située sur la membrane mitochondriale externe, où elle est essentielle à la fusion mitochondriale, un processus vital pour maintenir l'intégrité du réseau mitochondrial, l'homéostasie énergétique et la survie cellulaire. MFN1 contient un domaine GTPase N-terminal permettant la liaison et l'hydrolyse du GTP, deux régions de répétition d'heptapeptides (HR1 et HR2) qui forment des structures enroulées-bobinées médiatisant la dimérisation et l'ancrage entre les mitochondries adjacentes, et deux hélices transmembranaires ancrant la protéine dans la membrane externe. La HR2 étendue facilite l'interaction directe avec MFN1 ou MFN2 sur les mitochondries voisines pour favoriser une fusion efficace. MFN1 travaille en étroite collaboration avec MFN2 et OPA1 pour préserver la morphologie mitochondriale, stabiliser l'ADN mitochondrial (ADNmt), réguler la production d'ATP, soutenir les voies d'apoptose et permettre l'adaptation métabolique en cas de stress. Elle favorise également la mitophagie, empêchant l'accumulation de mitochondries dysfonctionnelles, et est importante pour l'embryogenèse et le développement neuronal. La perte ou le dysfonctionnement de MFN1 altère la dynamique mitochondriale, entraînant des déficits énergétiques, une neurodégénérescence et des maladies métaboliques. Bien que les mutations de MFN1 soient moins fréquentes que celles de MFN2, une déficience exacerbe la neuropathie de type Charcot-Marie-Tooth de type 2A, l'atrophie musculaire et les affections neuroinflammatoires ; une expression altérée de MFN1 est observée dans des maladies telles que Parkinson, Alzheimer et certains cancers.

Informations dutilisation

Application

WB, IP

Dilution

WB	IP
1:1000	1:30

Réactivité

Mouse, Rat

Source

Rabbit Monoclonal Antibody

MW

84 kDa

Tampon de stockage

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

Stockage
(À partir de la date de réception)

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

Méthodes expérimentales
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.

Méthodes expérimentales

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.

Références

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

Données dapplication

WB

Validé par Selleck

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