Proteins were isolated from microdissected formalin‐fixed, paraffin‐embedded (FFPE) cerebellum sections using the Qproteome FFPE Tissue kit (Qiagen, Valencia, CA) according to the manufacturers' instructions. Subsequent Western blotting of protein lysates and densitometric analysis were carried out as previously described.29 Primary antibodies used were anti‐β actin (ab8227, 1:5,000, Abcam), anti‐catalase (C0979, 1:2,000, Sigma‐Aldrich), anti‐human frataxin (ab110328, 1:2,000, Abcam), anti‐frataxin (ab113691, 1:2,000, Abcam), anti–glutathione peroxidase‐1 (ab22604, 1:1,000, Abcam), anti‐NeuN (ab177487, 1:5,000, Abcam), anti–nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2; sc‐722, 1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti‐peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1alpha) (sc‐13067, 1:3,000, Santa Cruz Biotechnology), anti‐SOD1 (ab16831, 1:5,000, Abcam), and anti‐SOD2 (ab16956, 1:5,000, Abcam). (Of note, increases in total frataxin are likely to be underestimated when compared to human frataxin due to differences in antibody (ab113691, Abcam) species reactivity; the human protein reactivity of the antibody is approximately 50% of mouse protein reactivity at the same concentration. Commercial mouse‐specific frataxin antibodies are not available.)
Ab113691
Manufactured by Abcam
Ab113691 is a lab equipment product offered by Abcam. It serves as a core functional component in research and laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Optima lc ms grade, by Thermo Fisher Scientific (4 mentions)
Ab175402, by Abcam (3 mentions)
Chemiluminescence reagent, by PerkinElmer (2 mentions)
Costar spin x centrifuge tube filter, by Corning (2 mentions)
Ab124680, by Abcam (2 mentions)
Ls c197243, by LifeSpan BioSciences (2 mentions)
Stainless steel beads for tissue homogenization, by Next Advance (2 mentions)
Anti mouse hrp, by Santa Cruz Biotechnology (2 mentions)
Anti rabbit hrp, by Santa Cruz Biotechnology (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Mab1594, by Merck Group (2 mentions)
Image lab software 5, by Bio-Rad (2 mentions)
Cell lysis buffer, by Cell Signaling Technology (2 mentions)
Pmsf protease inhibitor, by Cell Signaling Technology (2 mentions)
Captureselect aav9 resin, by Thermo Fisher Scientific (2 mentions)
Trypsin endoproteinase sequencing grade, by Promega (2 mentions)
Dynabeads protein g for immunoprecipitation, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Easypack protease inhibitor cocktail tablets, by Merck Group (2 mentions)
11 protocols using ab113691
1
Protein Extraction from FFPE Cerebellum
2
Frataxin Isoform Immunoblotting Analysis
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The His-frataxin-E standard (2 ng), His-frataxin-M standard (2 ng) and a portion from each whole blood eluate (20 μL) were mixed separately with 5 μL of NuPAGE LDS sample buffer (4X) containing 8% BME. The samples were then heated to 95 °C for 10 min before loading on a 12% NuPAGE Bis–Tris protein gel. NuPAGE MES SDS buffer was used for optimal separation of proteins in the 10–30 kDa range. The gel was run under 150 V for 1.5 h until the blue dye ran to the bottom of the gel. The proteins were transferred to a nitrocellulose membrane using the iBlot 2 gel transfer device and an iBlot 2 transfer stack. The membrane was probed with either an Abcam (Ab113691) anti-human frataxin mouse mAb (diluted 1:1000 with 5% milk in PBS containing 0.1% Tween-20) or an anti-human frataxin isoform E mAb generated by GenScript (diluted 1:1000 with 5% milk in PBS containing 0.1% Tween-20). A goat anti-rabbit HRP IgG (diluted 1:5000) was used as the secondary antibody for chemiluminescence detection. Chemiluminescence was generated using a 1:1 mixture of SuperSignal West femto stable peroxide buffer and luminol enhancer solution. Western blot images were captured on an ImageQuant LAS 4000 (GE Healthcare, Piscataway, NJ).
3
Frataxin Protein Detection Protocols
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Four protein standards were used in this study. Cynomolgus monkey mature frataxin (aa 81–210) was purchased from LifeSpan BioSciences (LS-G21788). Mouse recombinant intermediate form of frataxin (aa 41–207) was obtained from LifeSpan BioSciences (LS-G14956). Human and mouse mature frataxin were prepared using a protocol described previously (Guo et al. Anal. Chem.). Five antibodies were tested here, including AB113691, AB175402, AB124680 obtained from Abcam (Cambridge, MA), MAB1594 from Millipore-Sigma (Billerica, MA), and LS-C197243 from LifeSpan BioSciences (Seattle, WA). Anti-rabbit HRP and anti-mouse HRP were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX). Stainless steel beads for tissue homogenization were purchased from Next Advance (Troy, NY). NuPAGE™ LDS sample buffer was from Thermo Scientific (Waltham, MA) and chemiluminescence reagent (NEL103E001) was purchased from Perkin Elmer (Waltham, WA). Costar® Spin-X centrifuge tube filters (0.22 μm cellulose acetate) were obtained from Corning (Corning, NY).
4
Frataxin Protein Expression Analysis
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Frozen brain tissues were lysed using cell lysis buffer (Cell Signaling Technology, MA, United States) supplemented with 400 μM PMSF protease inhibitor (Cell Signaling Technology, MA, United States) and incubated on ice for 30 min. Lysates were subjected to centrifugation at 12,000 × g for 30 min at 4°C and protein concentrations were determined by Pierce BCA assay (Thermo Fisher Scientific, MA, United States). A total of 50 μg of protein lysates were boiled for 10 min and subjected to SDS-PAGE electrophoresis using 4–15% precast gels (Bio-Rad). Densitometry was calculated using the Image Lab Software 5.2.1 (Bio-Rad Laboratories, CA, United States). Antibodies used in this study are as follows: anti-Frataxin (1:250) (Abcam Cat# ab113691, RRID:AB_10862125 ), anti-Tubulin (1:10,000) (Abcam Cat# ab6160, RRID:AB_305328 ), rabbit anti-Rat HRP (1:5000) (Abcam Cat# ab6734, RRID:AB_955450 ), and goat anti-Mouse HRP (1:300) (Agilent Cat# P0447, RRID:AB_2617137 ). Non-relevant gel lanes and unspecific bands were excised by digital treatment using Power Point. Original uncropped images are presented as Supplementary material .
5
Frataxin Protein Detection Protocols
6
Quantitative Frataxin Immunoprecipitation
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All reagents and solvents were LC-MS grade quality unless otherwise noted. [13C615N2]-lysine and [13C615N1]-leucine were from Cambridge Isotope Laboratories (Andover, MA, USA). Anti-frataxin mouse mAb (clone 1D9) for cross-linking to magnetic beads was from LifeSpan Biosciences, Inc. (Seattle, WA), and the anti-frataxin mouse mAb for western blot analysis was 17A11 from Abcam (Cambridge, MA). LifeSpan Biosciences has discontinued clone 1D9. However, Abcam anti-frataxin rabbit polyclonal Ab175402 or mouse monoclonal Ab113691 can be used as alternatives in the IP procedure. EDTA-free protease inhibitor cocktail, DL-dithiothreitol (DTT), dimethyl pimelimidate dihydrochloride (DMP) were purchased from MilliporeSigma (Billerica, MA). LC grade water and acetonitrile were from Burdick and Jackson (Muskegon, MI, USA). Protein G magnetic beads were obtained from Life Technologies Corporation (Grand Island, NY). The heavy isotope leucine labeled AQUA peptide (Acetyl-MNL*RKSGTL*GHPGSL* with L* = [13C615N1]-L) was synthesized by Thermo Scientific (isotopic enrichment > 99%, HeavyPeptide AQUA custom synthesis service (Rockford, IL, USA).
7
Antibody Cross-Linking for Protein Pulldown
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Mouse
antifrataxin mAb (Abcam 17A11, Ab113691) was cross-linked to protein
G beads through DMP as described.20 (link) Briefly,
the mAb (4 μg) was first incubated with protein G magnetic Dynabeads
(0.5 mg) overnight at 4 °C to form the antibody-coupled beads.
The mAb-coupled beads were incubated with 13 mg/mL DMP solution for
1 h at room temperature to form the stable cross-linked antifrataxin
protein G magnetic Dynabeads. The cross-linked protein G beads can
be kept in phosphate-buffered saline (PBS) at 4 °C for a week.
antifrataxin mAb (Abcam 17A11, Ab113691) was cross-linked to protein
G beads through DMP as described.20 (link) Briefly,
the mAb (4 μg) was first incubated with protein G magnetic Dynabeads
(0.5 mg) overnight at 4 °C to form the antibody-coupled beads.
The mAb-coupled beads were incubated with 13 mg/mL DMP solution for
1 h at room temperature to form the stable cross-linked antifrataxin
protein G magnetic Dynabeads. The cross-linked protein G beads can
be kept in phosphate-buffered saline (PBS) at 4 °C for a week.
8
Immunofluorescence Imaging of Frataxin Knockdown
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To visualize the FxnKD we have used immunofluorescence (Abeti et al., 2015 (link)). Briefly, cells were fixed with 4% PFA, permeabilised with 0.5% Triton-X and incubated over night with an anti-frataxin antibody [17A11] (Abcam, ab113691; 1 μg/ml), and detected with a secondary antibody Alexa Fluor 568 goat anti-mouse (Thermo Fisher Scientific), diluted 1:1000. Nuclei were detected loading the cells with 300 nM DAPI for 5 min. Images were obtained using a Zeiss 710 LSM equipped with a META detection system and a 40x oil immersion objective.
9
Immunoprecipitation and Mass Spectrometry of Frataxin
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Protein G Dynabeads for immunoprecipitation, RIPA lysis buffer with EDTA, formic acid Optima LC/MS Grade, and CaptureSelect™ AAV9 resin were from ThermoFisher Scientific (Waltham, MA). Phosphate-buffered saline (PBS) ammonium bicarbonate (NH4HCO3), dimethyl pimelimidate dihydrochloride (DMP), ethanolamine, triethylamine (TEA), 0.2 M cross-linking buffer, pH 8.0, dithiothreitol (DTT), acetic acid (glacial), Tween-20, EDTA-free Easypack protease inhibitor cocktail tablets, bovine serum albumin (BSA), and sodium dodecyl sulfate (SDS) were from MilliporeSigma (Billerica, MA). Trypsin endoproteinase sequencing grade was from Promega (Madison, WI), acetonitrile (ACN), LC/MS grade water and Optima LC/MS Grade, were from Fisher Scientific (Pittsburgh PA). Mouse anti-frataxin monoclonal antibody (mAb) Ab113691 raised against the N-terminal sequence of hFXN was from Abcam (Walton, MA). hFXN-M protein and SILAC-hFXN-M protein internal standard were prepared as previously described43 (link). De-identified fresh frozen control (non-FRDA) heart tissue was supplied by BioIVT (Hicksville, NY) from consented donors and the relevant IRB approval was obtained. Subjects had typically died from heart failure.
10
Frataxin Expression Modulation by Epigenetic Inhibitors
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Cells were treated with 100 nM BIX01294, 2 μM GSK126 or combination of both for 72 h. Cells were washed with ice-cold PBS and lysed on ice for 30 min with cell lysis buffer (Cell Signaling Technology, 9803) supplemented with 400 μM PMSF protease inhibitor (Cell Signaling Technology, 8553). Lysates were subjected to centrifugation at 12,000 g for 30 min at 4°C and protein concentrations were determined using the BCA protein assay reagent (Thermo Scientific). 50 μg of protein lysates were boiled for 10 min and subjected to SDS-PAGE electrophoresis using 4–15% precast gels (Bio-Rad, 567-1084). Densitometry was calculated using the Image Lab Software 5.2.1 (Bio-Rad). Antibodies used in this study are as follows: anti-frataxin (1:250, ab113691, Abcam), β-actin (1:1000, A2066, Sigma), goat anti-Rabbit HRP (1:5000, P0448, Dako) and goat anti-Mouse HRP (1:5000, P0447, Dako).
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