Ab110324

Manufactured by Abcam

Sourced in United States, United Kingdom

Ab110324 is a laboratory equipment product designed for scientific research purposes. It serves as a core function, without additional interpretation or extrapolation on its intended use.

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14 protocols using ab110324

Mitochondrial Protein Immunoblotting

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Antibodies used in the experiments were anti-CypD (1:1000, ab110324, Abcam, Cambridge, MA, United States); anti-VDAC1 (1:1000, ab154856, Abcam); anti-ANT (1:1000, ab180715, Abcam); and anti-β-Actin (1:10,000, Sigma, St. Louis, MO, United States). See Supplementary Material for details.

Zhang Y., Lu P., Liang F., Liufu N., Dong Y., Zheng J.C, & Xie Z. (2020). Cyclophilin D Contributes to Anesthesia Neurotoxicity in the Developing Brain. Frontiers in Cell and Developmental Biology, 7, 396.

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Quantitative Analysis of CypD Expression

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Tg CypD+, CypD−, and nonTg mouse brains were fixed by perfusion with 4% paraformaldehyde and coronal sections (30 μm) were cut with a Vibratome (Leica VTS1000, Wetzlar Germany). Sections were collected and immersed in wash buffer (0.1 M sodium phosphate, 0.5 M sodium chloride, 0.1% Triton X-100, pH 7.4) for 1 hour. After preincubation with blocking solution (10% normal goat serum, 0.3% Triton X-100 in PBS , pH 7.4) for one hour at room temperature, sections were co-immunostained with primary antibodies [mouse anti-CypD (Ab110324, 1:500; Abcam) and rabbit anti-SODII (1:5000, ADI-SOD-111, Enzo life Sciences, USA) or rabbit anti-MAP2 (1 : 500, A16657, life technologies, USA) at 4 °C for overnight. Sections were then incubated with Alexa Fluor 594-conjugated goat anti-rabbit IgG and 488 goat anti-mouse IgG secondary antibodies (1 : 1000, Invitrogen) for 1 h at room temperature. Nuclei were stained by DRAQ5 (5 μM, Cell Signaling) for 10 min at room temperature. The staining images were taken under confocal microscopy. Brain sections incubated with non-immune IgG or 2^nd antibody alone were used as negative controls. An investigator who was blinded to experimental groups analyzed all images. For quantification of human CypD staining, brain sections were randomly selected and measured using MetaMorph software (Molecular Devices, CA).

Zhang Z., Wang Y., Yan S., Du F, & Yan S.S. (2015). NR2B-dependent Cyclophilin D translocation suppresses the recovery of synaptic transmission after oxygen-glucose deprivation. Biochimica et biophysica acta, 1852(10 Pt A), 2225-2234.

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Western Blot Analysis of CypD and DCX

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Western blot analysis was performed as described [42 (link)]. Specifically, anti-CypD (1:1000, ab110324, Abcam) and anti-DCX (1:500, ab113435, Abcam) were used to detect CypD and DCX separately. β-actin antibody (1:5000, 42 kDa, Sigma, St. Louis, MO, USA) was used to detect β-actin as a loading control. Quantification of Western blots was performed as described previously [42 (link)]. A protein levels of 100% indicates control levels. Signal intensity was analyzed using a Bio-Rad (Hercules, CA, USA) image program. The Western blots were quantified using two steps. First, β-actin levels were used to normalize (e.g., determining the ratio of CypD levels to β-actin levels) for any loading differences in total protein amounts. Second, changes in CypD levels in treated cells were presented as percentages of the corresponding levels in the control cells. Each band in the Western blot represented an independent experiment. The results were averaged by six independent experiments.

Lu P., Liang F., Dong Y., Xie Z, & Zhang Y. (2023). Sevoflurane Induces a Cyclophilin D-Dependent Decrease of Neural Progenitor Cells Migration. International Journal of Molecular Sciences, 24(7), 6746.

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Evaluating Mitochondrial Protein Levels

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Anti-ANT (adenine nucleotide translocase; detects isoforms 1,2 and 3, sc-9300 from Santa Cruz); anti-ATP5A (α- subunit, ab14748, Abcam); anti-ATP5G (c-subunit, ab 180149, Abcam) anti-ATP synthase immunocapture antibody (ab109867 Abcam); anti-complex 1 immunocapture antibody (ab109798, Abcam); anti-complex 3 immunocapture antibody (ab109862, Abcam); anti-CypD (cyclophilin D, ab 110324, Abcam); anti-GAPDH (MAB374 Chemicon) anti-mtCK (mitochondrial creatine kinase, sc-15169, Santa Cruz); anti-NDUFAB1 (ab96230, Abcam); anti-OSCP (oligomycin sensitivity conferring protein, sc-365162, Santa Cruz); and anti-VDAC (voltage dependent anion channel)/anti-porin 31HL (529534, Calbiochem).

Beutner G., Alanzalon R.E, & Porter GA J.r. (2017). Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes. Scientific Reports, 7, 14488.

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Immunofluorescence Analysis of Neural Progenitor Cells

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The cultured cells were seeded onto six-well plates, and their differentiation potency was characterized and tested using immunofluorescence staining analysis. To fix the cells, 4% paraformaldehyde (PFA) was used. Mouse monoclonal anti-nestin antibody (1:500, Abcam, Boston, MA, USA), rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (1:500, Abcam), and mouse monoclonal anti-β-tubulin III antibody (1:500, Abcam) were used for NPCs identification. D4′, 6′-diamidion-2′-phenylindole (DAPI, 1:1000, Abcam) was used to counterstain the nuclei. To detect CypD and Complex V (mitochondrial marker), anti-CypD (1:200, ab110324, Abcam) and anti-complex V (1:200, 45, 9000, Invitrogen, Carlsbad, CA, USA) antibodies were used. After fixing the cells for 20 min, they were placed in 0.1% Triton X-100 for 30 min, followed by a blocking buffer (10% goat serum) for 1 h. The cells were then incubated with primary antibodies overnight at 4 °C. Samples were incubated in secondary antibodies [fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated IgG] for 2 h, followed by DAPI incubation for 5 min at room temperature. The slides were rinsed with DPBS, and the images were captured using a laser confocal microscope (TSC SP2, Leica, Manheim, Germany).

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Protein Expression and Apoptosis Profiling

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Equal amounts of protein extracts were separated by 20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to 0.22 μm or 0.45 μm polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature before being incubated with primary antibodies overnight at 4°C, which included antibodies against cyclophilin F (ab110324; Abcam PLC), Beclin1 (ab217179; Abcam PLC), TOMM20 (ab186735; Abcam PLC), caspase‐3 (19677‐1‐AP; ProteinteCh Group, Inc.) and cleaved caspase‐3 (9664; Cell Signaling Technology, Inc.). The next day, the secondary antibody was incubated for an hour with the corresponding horseradish peroxidase‐conjugated mouse or rabbit antibody (1:5000). Immunoreactive proteins were then measured through an enhanced chemiluminescence detection system (GE AI680; General Electric Company).

Yang M., Wang Z., Xie J., Reyad‐ul‐Ferdous M., Li S, & Song Y. (2023). Cyclophilin D as a potential therapeutic target of liver ischemia/reperfusion injury by mediating crosstalk between apoptosis and autophagy. Chronic Diseases and Translational Medicine, 9(3), 238-249.

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Spermatozoa Protein Extraction and Analysis

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Spermatozoa were homogenized in RIPA lysis buffer (WSE-7420; ATTO, Tokyo, Japan) containing 1% (v/v) protease and phosphatase inhibitor cocktail. After five rounds of sonication for 5 s at 4 °C, protein samples were obtained by centrifugation at 18,000× g for 20 min, and supernatants were subjected to western blot. Antibodies specific for rabbit anti GSK3α/β (#5676; Cell Signaling, Beverly, MA, USA), rabbit anti p-GSK3α/β (Ser21/9) (#9327; Cell Signaling), mouse anti cyclophilin D (ab110324; Abcam, Cambridge, UK), rabbit anti ATP synthase F1 subunit alpha (ATP5A) (ab176569; Abcam), rabbit anti β-tubulin (ab108342; Abcam), and rabbit anti GAPDH (sc-25778; SantaCruz Biotechnology, Dallas, TX, USA) were mixed in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with the membranes overnight at 4 °C. After being rinsed three times with TBST for 20 min, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG (ab6721; Abcam) and anti-mouse IgG (ab6728; Abcam) mixed with skim milk in TBST for 1 h. After washing three times with TBST for 20 min, the signals were detected using ECL Reagent (RPN2232; Amersham Bioscience) and Fusion SL (Vilber Lourmat, Marne-la-Vallée, France). The relative band intensities were analyzed using ImageJ (Ver.1.51j8; National Institutes of Health) and are expressed in arbitrary units (AU).

Park S.H, & Gye M.C. (2024). Inhibition of mitochondrial cyclophilin D, a downstream target of glycogen synthase kinase 3α, improves sperm motility. Reproductive Biology and Endocrinology : RB&E, 22, 15.

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Quantitative Protein Analysis in Cell Lysates

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The cells were collected and lysed for 1 h on ice in a strong RIPA buffer containing a protease inhibitor cocktail (Merk, 539134). The samples were subjected to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to PVDF (0.45 µm) membranes. The anti-ARC antibodies (1:2000) were from Abcam (ab126238) and Santa Cruz (sc-374177). The anti-CypD antibodies (1:2000) were from Abcam (ab110324). The anti-p53 antibody (1:1000) was from Cell Signaling Technology (mAb #2524). The anti-α-actin antibody was from Santa Cruz Biotechnology. The anti-myc antibody was from Biorbyt, China. The anti-HA antibody was from Santa Cruz Biotechnology. Primary antibody was incubated at 4 °C overnight. After washing with PBS-Tween 20 five times, HRP-conjugated secondary antibodies were incubated at room temperature for 1 h. Bands were quantitated using Image J and α-actin was used as the loading control. Fold change was normalized to the indicated control.

Xu T., Ding W., Ao X., Chu X., Wan Q., Wang Y., Xiao D., Yu W., Li M., Yu F, & Wang J. (2018). ARC regulates programmed necrosis and myocardial ischemia/reperfusion injury through the inhibition of mPTP opening. Redox Biology, 20, 414-426.

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Comprehensive Mitochondrial Protein Analysis

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For Western blot analysis, the following antibodies were used in a 1:1000 dilution: anti-β (ab14730, Abcam), anti-γ (PA5-29975, ThermoFisher), anti-b (ab117991, Abcam), anti-OSCP (ab110276, Abcam), anti-f (ab200715, Abcam), anti-g (ab126181, Abcam), anti-e (ab122241, Abcam), anti-c (ab181243, Abcam), anti-a (ab192423, Abcam), anti-citrate synthase (ab96600, Abcam), anti-vinculin (V4505, Sigma), anti-prohibitin (MS-261-P1, NeoMarkers), anti-GAPDH (2118, Cell Signaling), anti-CyPD (ab110324, Abcam), anti-ANT2 (14671, Cell Signaling), anti-ANT3 (PA5-35113, ThermoFischer), anti-OXPHOS (ab110411, Abcam), anti-HKII (sc130358, Santa Cruz), anti-UQCRC1 (sc65238, Santa Cruz), anti-Grim19 (sc271013, Santa Cruz), and anti-SDHA (sc166947, Santa Cruz). Statistical comparison of data was assessed with the two-sided Student’s t-test.

Carrer A., Tommasin L., Šileikytė J., Ciscato F., Filadi R., Urbani A., Forte M., Rasola A., Szabò I., Carraro M, & Bernardi P. (2021). Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase. Nature Communications, 12, 4835.

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Mitochondrial protein analysis protocol

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Mitochondria were lysed in PBS containing 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with 5 mmol L⁻¹ EDTA, 1 mmol L⁻¹ Na₃VO₄, 20 mmol L⁻¹ NaF, 1 mmol L⁻¹ DTT, and protease inhibitor cocktail (Sigma P2714). Proteins were separated by SDS‐10% PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, and incubated overnight with primary antibodies. Primary antibodies used were OXPHOS (Abcam, ab110413), ANT2 (Santa Cruz, SC‐9299), UCP2 (BioLegend, 615902), and actine (Sigma A5060).
For cyclophilinD (CypD: Ppif) content determination, samples were frozen and heated five times and then centrifuged at 100,000 × g for 3 h. Supernatant were collected and considered as the matrix content. Pellets were washed with PBS, centrifuged at 100,000 × g for 3 h and proteins were extracted with PBS containing 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS supplemented with 5 mmol L⁻¹ EDTA, 1 mmol L⁻¹ Na₃VO₄, 20 mmol L⁻¹ NaF, 1 mmol L⁻¹ DTT, and protease inhibitor cocktail (Sigma P2714). 12 μg of proteins was separated by SDS‐15% PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, and incubated overnight with primary CypD antibodies (Abcam, ab110324). Signals were detected with a horseradish peroxidase‐conjugated secondary antibody (Biorad, 172‐10‐19) and revealed with an enhanced chemiluminescence system (Pierce).

Vial G., Le Guen M., Lamarche F., Detaille D., Cottet‐Rousselle C., Demaison L., Hininger‐Favier I., Theurey P., Crouzier D., Debouzy J., Dubouchaud H, & Fontaine É. (2016). Liver mitochondrial function in ZDF rats during the early stages of diabetes disease. Physiological Reports, 4(3), e12686.

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