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Anti-POLG Rabbit Antibody [M21J18]

Catalog No.: F2171

Application: Reactivity: Mouse, Rat, Human

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Exposure time of at least 120s is recommended.

Usage Information

Dilution
WB1:1000 - 1:10000

Application
WB

Reactivity
Mouse, Rat, Human

Source
Rabbit

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

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
140 kDa 140 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight.

Positive Control	HEK-293T; HepG2; RAW 264.7; C6; MCF7; 293T
Negative Control

Exprimental Methods

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)

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)

Datasheet & SDS

Biological Description

Specificity
POLG Rabbit mAb detects endogenous levels of total POLG protein.

Subcellular Location
Mitochondrion, Mitochondrion nucleoid

Uniprot ID
P54098

Clone
M21J18

Synonym(s)
DNA polymerase subunit gamma-1, Mitochondrial DNA polymerase catalytic subunit, PolG-alpha, POLGA, POLG1, MDP1, POLG.

Background
Mitochondrial DNA (mtDNA) resides in discrete nucleoids within the inner matrix of the mitochondrion, with each nucleoid typically containing one or two copies of mtDNA. Replication of mtDNA is carried out by a specialized replisome composed of core replication proteins, including DNA polymerase γ (pol γ), the mitochondrial single-stranded DNA-binding protein, the Twinkle mtDNA helicase, as well as topoisomerases and RNase H. Human pol γ is a heterodimer consisting of two subunits:POLG, the 140 kDa catalytic subunit encoded by the POLG gene at chromosomal locus 15q25, possesses DNA polymerase, 3′→5′ exonuclease, and 5′-deoxyribose phosphate (5′-dRP) lyase activities. It has an amino-terminal exonuclease domain connected by a linker to the carboxy-terminal polymerase domain. POLG2, the 55 kDa accessory subunit encoded by POLG2 at chromosomal locus 17q24, forms a dimer and enhances polymerase processivity by increasing the catalytic subunit’s affinity for DNA. Mutations in POLG are a major cause of inherited mitochondrial disorders, with up to 2% of the population carrying pathogenic variants. These mutations can lead to mtDNA depletion syndromes in early childhood or to later-onset conditions resulting from mtDNA deletions. POLG-related disorders form a continuum of overlapping phenotypes, with onset ranging from infancy to late adulthood. The six primary clinical syndromes linked to POLG mutations are: Alpers–Huttenlocher syndrome (one of the most severe phenotypes), Childhood myocerebrohepatopathy spectrum, Myoclonic epilepsy myopathy sensory ataxia, Ataxia neuropathy spectrum, Autosomal recessive progressive external ophthalmoplegia, Autosomal dominant progressive external ophthalmoplegia. , ,

Background

Mitochondrial DNA (mtDNA) resides in discrete nucleoids within the inner matrix of the mitochondrion, with each nucleoid typically containing one or two copies of mtDNA. Replication of mtDNA is carried out by a specialized replisome composed of core replication proteins, including DNA polymerase γ (pol γ), the mitochondrial single-stranded DNA-binding protein, the Twinkle mtDNA helicase, as well as topoisomerases and RNase H. Human pol γ is a heterodimer consisting of two subunits:POLG, the 140 kDa catalytic subunit encoded by the POLG gene at chromosomal locus 15q25, possesses DNA polymerase, 3′→5′ exonuclease, and 5′-deoxyribose phosphate (5′-dRP) lyase activities. It has an amino-terminal exonuclease domain connected by a linker to the carboxy-terminal polymerase domain. POLG2, the 55 kDa accessory subunit encoded by POLG2 at chromosomal locus 17q24, forms a dimer and enhances polymerase processivity by increasing the catalytic subunit’s affinity for DNA. Mutations in POLG are a major cause of inherited mitochondrial disorders, with up to 2% of the population carrying pathogenic variants. These mutations can lead to mtDNA depletion syndromes in early childhood or to later-onset conditions resulting from mtDNA deletions. POLG-related disorders form a continuum of overlapping phenotypes, with onset ranging from infancy to late adulthood. The six primary clinical syndromes linked to POLG mutations are: Alpers–Huttenlocher syndrome (one of the most severe phenotypes), Childhood myocerebrohepatopathy spectrum, Myoclonic epilepsy myopathy sensory ataxia, Ataxia neuropathy spectrum, Autosomal recessive progressive external ophthalmoplegia, Autosomal dominant progressive external ophthalmoplegia. , ,

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

Reference Table for Selecting PVDF Membrane Pore Size Specifications

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%