Datenblatt

Biologische Beschreibung

Spezifität	POLG Antibody [M21J18] weist endogene Spiegel des gesamten POLG-Proteins nach.
Hintergrund	Mitochondriale DNA (mtDNA) befindet sich in diskreten Nukleoiden innerhalb der inneren Matrix des Mitochondriums, wobei jeder Nukleoid typischerweise eine oder zwei Kopien von mtDNA enthält. Die Replikation von mtDNA wird von einem spezialisierten Replisom durchgeführt, das aus Kernreplikationsproteinen besteht, einschließlich DNA-Polymerase γ (pol γ), dem mitochondrialen einzelsträngigen DNA-Bindungsprotein, der Twinkle mtDNA-Helikase sowie Topoisomerasen und RNase H. Die menschliche pol γ ist ein Heterodimer, das aus zwei Untereinheiten besteht: POLG, die 140 kDa katalytische Untereinheit, die durch das POLG-Gen am Chromosomenlocus 15q25 kodiert wird, besitzt DNA-Polymerase-, 3′→5′-Exonuklease- und 5′-Desoxyribosephosphat- (5′-dRP) Lyase-Aktivitäten. Sie hat eine amino-terminale Exonuklease-Domäne, die durch einen Linker mit der carboxy-terminalen Polymerase-Domäne verbunden ist. POLG2, die 55 kDa akzessorische Untereinheit, die durch POLG2 am Chromosomenlocus 17q24 kodiert wird, bildet ein Dimer und erhöht die Polymerase-Prozessivität, indem sie die Affinität der katalytischen Untereinheit zur DNA erhöht. Mutationen in POLG sind eine Hauptursache für vererbte mitochondriale Erkrankungen, wobei bis zu 2 % der Bevölkerung pathogene Varianten tragen. Diese Mutationen können zu mtDNA-Depletionssyndromen im frühen Kindesalter oder zu später auftretenden Erkrankungen, die aus mtDNA-Deletionen resultieren, führen. POLG-bedingte Erkrankungen bilden ein Kontinuum überlappender Phänotypen mit einem Beginn, der vom Säuglingsalter bis ins späte Erwachsenenalter reicht. Die sechs primären klinischen Syndrome, die mit POLG-Mutationen verbunden sind, sind: Alpers–Huttenlocher-Syndrom (einer der schwersten Phänotypen), myocerebrohepatisches Spektrum im Kindesalter, myoklonische Epilepsie Myopathie sensorische Ataxie, Ataxie-Neuropathie-Spektrum, autosomal-rezessive progressive externe Ophthalmoplegie, autosomal-dominante progressive externe Ophthalmoplegie.

Spezifität

POLG Antibody [M21J18] weist endogene Spiegel des gesamten POLG-Proteins nach.

Hintergrund

Mitochondriale DNA (mtDNA) befindet sich in diskreten Nukleoiden innerhalb der inneren Matrix des Mitochondriums, wobei jeder Nukleoid typischerweise eine oder zwei Kopien von mtDNA enthält. Die Replikation von mtDNA wird von einem spezialisierten Replisom durchgeführt, das aus Kernreplikationsproteinen besteht, einschließlich DNA-Polymerase γ (pol γ), dem mitochondrialen einzelsträngigen DNA-Bindungsprotein, der Twinkle mtDNA-Helikase sowie Topoisomerasen und RNase H. Die menschliche pol γ ist ein Heterodimer, das aus zwei Untereinheiten besteht: POLG, die 140 kDa katalytische Untereinheit, die durch das POLG-Gen am Chromosomenlocus 15q25 kodiert wird, besitzt DNA-Polymerase-, 3′→5′-Exonuklease- und 5′-Desoxyribosephosphat- (5′-dRP) Lyase-Aktivitäten. Sie hat eine amino-terminale Exonuklease-Domäne, die durch einen Linker mit der carboxy-terminalen Polymerase-Domäne verbunden ist. POLG2, die 55 kDa akzessorische Untereinheit, die durch POLG2 am Chromosomenlocus 17q24 kodiert wird, bildet ein Dimer und erhöht die Polymerase-Prozessivität, indem sie die Affinität der katalytischen Untereinheit zur DNA erhöht. Mutationen in POLG sind eine Hauptursache für vererbte mitochondriale Erkrankungen, wobei bis zu 2 % der Bevölkerung pathogene Varianten tragen. Diese Mutationen können zu mtDNA-Depletionssyndromen im frühen Kindesalter oder zu später auftretenden Erkrankungen, die aus mtDNA-Deletionen resultieren, führen. POLG-bedingte Erkrankungen bilden ein Kontinuum überlappender Phänotypen mit einem Beginn, der vom Säuglingsalter bis ins späte Erwachsenenalter reicht. Die sechs primären klinischen Syndrome, die mit POLG-Mutationen verbunden sind, sind: Alpers–Huttenlocher-Syndrom (einer der schwersten Phänotypen), myocerebrohepatisches Spektrum im Kindesalter, myoklonische Epilepsie Myopathie sensorische Ataxie, Ataxie-Neuropathie-Spektrum, autosomal-rezessive progressive externe Ophthalmoplegie, autosomal-dominante progressive externe Ophthalmoplegie.

Nutzungsinformationen

Anwendung

WB

Verdünnung

WB

1:1000 - 1:10000

Reaktivität

Mouse, Rat, Human

Quelle

Rabbit Monoclonal Antibody

MW

140 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

Experimentelle Methoden
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA 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 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 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: 5%. 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. (Exposure time of at least 120s is recommended)

Experimentelle Methoden

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA 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 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 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: 5%. 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. (Exposure time of at least 120s is recommended)

Referenzen

https://pubmed.ncbi.nlm.nih.gov/30451971/

Anwendungsdaten

WB

Validiert von Selleck

Lane 1: HEK-293T, Lane 2: HepG2