C10orf2 antibody (Middle Region)

Catalog No. ABIN2775251

This independently validated Rabbit Polyclonal antibody specifically detects C10orf2 in WB. It exhibits reactivity toward Human, Mouse, Rat, Cow, Guinea Pig, Horse, Dog, Rabbit and Zebrafish (Danio rerio). It has been mentioned in 2+ publications

Quick Overview for C10orf2 antibody (Middle Region) (ABIN2775251)

Target: C10orf2 (C10ORF2) (Chromosome 10 Open Reading Frame 2 (C10ORF2))

Reactivity: Human, Mouse, Rat, Cow, Guinea Pig, Horse, Dog, Rabbit, Zebrafish (Danio rerio)

Host: Rabbit

Clonality: Polyclonal

Conjugate: This C10orf2 antibody is un-conjugated

Application: Western Blotting (WB)

Product Details anti-C10orf2 Antibody

Binding Specificity: Middle Region

Purpose: TWNK Antibody - middle region

Sequence: GVFRKFATDN NCHVTLVIHP RKEDDDKELQ TASIFGSAKA SQEADNVLIL

Predicted Reactivity: Cow: 100%, Dog: 100%, Guinea Pig: 100%, Horse: 100%, Human: 100%, Mouse: 100%, Rabbit: 100%, Rat: 100%, Zebrafish: 93%

Characteristics: This is a rabbit polyclonal antibody against PEO1. It was validated on Western Blot using a cell lysate as a positive control.

Purification: Affinity Purified

Immunogen: The immunogen is a synthetic peptide directed towards the middle region of human PEO1

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

We also sell a specific blocking peptide that can be used in combination with this antibody. You can find the blocking peptide under AAP36483-100UG

Restrictions: For Research Use only

Independent Validation (WB)

Validation #100038 (Western Blotting)

by: Mitochondrial Biology, University of Eastern Finland

No.: #100038

Date: 08/08/2016

Antigen: PEO1 antibody - middle region

Lot Number: QC6322

Method validated: Western Blotting

Positive Control: HeLa cells, HeLa cells overexpressing PEO1

Negative Control: rho0 HeLa cells

Notes: Passed, regarding sensitivity and specificity. The antibody detects an antigen at the expected apparent molecular weight of its antigen.

Validation Images

75µg total protein of HeLa cells overexpressing PEO1 (lane 1), HeLa cells (lane 2), and rho0 Hela cells (lane 3) were separated on an denaturing 8% polyacrylamid gel. Bands were revealed as described in the protocol sections with ABIN2775251 diluted 1:1000.

Full Methods

Primary Antibody: ABIN2775251

Secondary Antibody: Goat-anti rabbit IgG (H+L), HRP-linked (Invitrogen, A-16104)

Full Protocol:

• Cell lysates were propared from HeLa cells, HeLa cells overexpressing PEO1, and rho⁰ HeLa cells.
• 75µg total protein or each sample were separated on a denaturing 8% SDS-PAGE gel (Laemmli 1970).
• Immunoblot onto supported nitrocellulose membrane (Protran, GE Healthcare No. 1060005, 0.2µm) (Towbin et al., 1979).
• Blocking of the membrane in 3% skim milk in TBST (50mM Tris-HCl, pH 7.4, 150mM NaCl, 0.1% Tween 20) for 1h at room temperature.
• Incubation with primary antibody ABIN2775251 diluted 1:1000 in 3% BSA in TBST at 4°C overnight.
• Washing in TBST for 3x 5min.
• Incubation with goat-anti rabbit IgG (H+L), HRP-linked (Invitrogen, A-16104) diluted 1:15000 in 3% BSA in TBST for 1h at room temperature.
• Washing in TBST for 5x 10min.
• Chemiluminescence detection with luminol solution (250µg/ml Na-Luminol, 0,01% H₂O₂, 11µg/ml p-hydroxy-coumaric acid in 100mM Tris pH 8.5) and image capture on Kodak ECL film.

Experimental Notes: PEO1 runs usually at 95-100 kDa, although the theoretical size is 72 kDa, often it is also visible as dimer.

Independent Validation (ICC)

Validation #100039 (Immunocytochemistry)

by: Mitochondrial Biology, University of Eastern Finland

No.: #100039

Date: 08/08/2016

Antigen: PEO1 antibody - middle region

Lot Number: QC6322

Method validated: Immunocytochemistry

Positive Control: Mouse-anti-ds DNA antibody (clone 35I9)

Notes: Passed, regarding sensitivity and specificity. The antibody shows a staining pattern consistent with the expected mitochondrial localization of PEO1.

Validation Images

ABIN2775251 was used at a 1:200 dilution to visualize PEO1 (green, secondary antibody AF488 conjugate). A dsDNA antibody (red, secondary antibody AF594 conjugate) and a DAPI counterstain (blue) were used as reference.

Full Methods

Primary Antibody: ABIN2775251

Secondary Antibody: Goat-anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (Invitrogen, A-11034, Lot 702323)

Full Protocol:

• HeLa cells were grown in DMEM high glucose + 10% FBS on acid-cleaned glass coverslips to 50% confluency.
• Cells were fixed in 4% para-formaldehyde in growth medium for 25min at RT, then washed once with growth medium and twice with PBS.
• Permeabilization was achieved by incubation with 0.5% Triton X-100, 10% fetal bovine serum in PBS for 15min.
• Primary antibodies:
• • ABIN2775251 diluted 1:200 in PBS with 0.1% Triton X-100 , 10% fetal bovine serum.
  • Co-staining of mitochondrial DNA with monoclonal antibody mouse-anti-dsDNA clone 35I9 DNA (Abcam ab27156, Lot GR27035-10) diluted 1:400 in PBS with 0.1% Triton X-100, 10% fetal bovine serum.
• Incubation for 1h at RT.
• Wash 3x 5min with PBS.
• Secondary antibodies:
• • Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (Invitrogen, A-11034, Lot 702323) diluted 1:1000 in PBS with 0,1% Triton X-100 , 10% fetal bovine serum, 1µM DAPI.
  • Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 594 conjugate (Invitrogen, A-11032, Lot 621333).
• Incubation for 1h at RT.
• Wash 3x 5min with PBS.
• The coverslips were mounted with Immu-mount mounting medium (Thermo Fisher, 9990402) and imaged with a 630 x magnification on a Zeiss Axioplan 2 fluorescent microscope using an Axiocam camera and Axiovision 4.8 software.
• For visualization of PEO1 the excitation wavelength was 488nm and emission wavelength >535nm, exposure time 53ms. DNA was visualized by excitation at 580nm and emission at >590nm, exposure time 410ms.

Handling

Format: Liquid

Concentration: 0.5 mg/mL

Buffer: Purified antibody supplied in 1x PBS buffer with 0.09 % (w/v) sodium azide and 2 % sucrose.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Handling Advice: prevent freeze-thaw cycles

Storage: 4 °C,-20 °C

Storage Comment: For short term use, store at 2-8C up to 1 week. For long term storage, store at -20 °C in small aliquots to prevent freeze-thaw cycles.

References for anti-C10orf2 antibody (ABIN2775251)

Wang, Xu, Alontaga, Chen, Liu: "Impaired p32 regulation caused by the lymphoma-prone RECQ4 mutation drives mitochondrial dysfunction." in: Cell reports, Vol. 7, Issue 3, pp. 848-58, (2014) (PubMed).

Ylikallio, Tyynismaa, Tsutsui, Ide, Suomalainen: "High mitochondrial DNA copy number has detrimental effects in mice." in: Human molecular genetics, Vol. 19, Issue 13, pp. 2695-705, (2010) (PubMed).

Target Details for C10orf2

Target: C10orf2 (C10ORF2) (Chromosome 10 Open Reading Frame 2 (C10ORF2))

Alternative Name: TWNK

Background:

Background Information: This gene encodes a hexameric DNA helicase which unwinds short stretches of double-stranded DNA in the 5' to 3' direction and, along with mitochondrial single-stranded DNA binding protein and mtDNA polymerase gamma, is thought to play a key role in mtDNA replication. The protein localizes to the mitochondrial matrix and mitochondrial nucleoids. Mutations in this gene cause infantile onset spinocerebellar ataxia (IOSCA) and progressive external ophthalmoplegia (PEO) and are also associated with several mitochondrial depletion syndromes. Alternative splicing results in multiple transcript variants encoding distinct isoforms.

Gene Name: twinkle mtDNA helicase

Alternative Symbols: PEO, PEO1, SCA8, ATXN8, IOSCA, PEOA3, SANDO, TWINL, MTDPS7, PRLTS5, C10orf2

Protein Name: twinkle protein, mitochondrial

Molecular Weight: 77 kDa

Gene ID: 56652

NCBI Accession: NP_068602

UniProt: Q96RR1