Mitofusin 2 d2d10

Manufactured by Cell Signaling Technology

Sourced in United States

Mitofusin-2 (D2D10) is a rabbit monoclonal antibody that recognizes the Mitofusin-2 protein. Mitofusin-2 is a GTPase that plays a key role in mitochondrial fusion.

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Lab products found in correlation

Tyrosine hydroxylase, by Merck Group (2 mentions) Enhanced chemiluminescence, by GE Healthcare (2 mentions) β tubulin, by Merck Group (2 mentions) Drp1 d6c7, by Cell Signaling Technology (2 mentions) Tom20 d8t4n, by Cell Signaling Technology (2 mentions) Disposable pellet mixers with pestle motor, by Avantor (2 mentions) Dopamine d2 receptor, by Merck Group (2 mentions) Phospho tau ser202 thr205 at8, by Thermo Fisher Scientific (2 mentions) Ha tag c29f4, by Cell Signaling Technology (2 mentions) β actin, by Abcam (2 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Image pro plus 6, by Media Cybernetics (1 mentions) α synuclein, by BD (1 mentions) Ripa buffer, by Bio-Rad (1 mentions) β actin d6a8, by Cell Signaling Technology (1 mentions) Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions) Supersignal west pico plus kit, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Chemidoc system, by Bio-Rad (1 mentions)

4 protocols using mitofusin 2 d2d10

Immunohistochemistry of Mitochondrial Dynamics

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Paraffin-embedded kidney sections (4 μm) were deparaffanized in xylenes, rehydrated in a graded series of ethanol (100%–75%), and washed in distilled water. Then, antigen retrieval was performed and sections were incubated in 3% H₂O₂ for 25 min. After blocking with 3% bovine serum albumin for 30 min at room temperature, sections were incubated in specific primary antibodies overnight at 4°C. Sections were washed with phosphate buffer (pH 7.4) three times and incubated with anti-goat secondary antibody for 50 min. After another three washes with phosphate buffer, the sections were visualized by incubating with diaminobenzidine, counterstained by hematoxylin, and dehydrated.
Immunohistochemistry was performed with antibodies for DRP1 (OTI4F6; Abcam, UK), OPA1 (EPR11057; Abcam), Mitofusin1 (3C9; Abcam), and Mitofusin2 (D2D10; Cell Signaling Technology, USA). And Image-Pro Plus 6.0 (Media Cybernetics, USA) was used to analyze image of Immunohistochemistry.

Long R.T., Peng J.B., Huang L.L., Jiang G.P., Liao Y.J., Sun H., Hu Y.D, & Liao X.H. (2019). Augmenter of Liver Regeneration Alleviates Renal Hypoxia-Reoxygenation Injury by Regulating Mitochondrial Dynamics in Renal Tubular Epithelial Cells. Molecules and Cells, 42(12), 893-905.

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Protein Extraction and Western Blot Analysis of Neural Tissues

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Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0 °C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500 μl, 5 μl aprotinin, PMSF 50 μl, Na₃VO₄ 100 μl and NaF 20 μl in 4.32 ml PBS. Extracts were clarified by centrifugation at 4 °C (13,200g for 20 min). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE [45 (link)]. The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), α-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.

Vanan S., Zeng X., Chia S.Y., Varnäs K., Jiang M., Zhang K., Saw W.T., Padmanabhan P., Yu W.P., Zhou Z.D., Halldin C., Gulyás B., Tan E.K, & Zeng L. (2020). Altered striatal dopamine levels in Parkinson’s disease VPS35 D620N mutant transgenic aged mice. Molecular Brain, 13, 164.

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Western Blot Analysis of Mitofusin-2

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Western blots were performed according to standard protocols. Briefly, cell lysis was conducted with RIPA buffer (+1% Triton-X100) (Bio-Rad formulation) supplemented with protease inhibitor cocktail (1:50; Sigma Aldrich, Vienna, Austria). Samples were frozen in liquid nitrogen and thawed for three times and incubated for 30 min on ice. Protein amounts were measured with Pierce BCA Protein Assay Kit (ThermoScientific, United States). 40 µg protein samples were loaded on a 12.5% SDS-PAGE gel together with PageRuler™ Plus Prestained Protein Ladder (Fisher Scientific, Vienna Austria). Blots were blocked (5% milk) and antibodies diluted in 5% milk in TBS-Tween. The following antibodies were used: Mitofusin-2 (D2D10, 1:1,000, Cell Signaling Technology, MA, United States), and β-Actin (D6A8, 1:1,000, Cell Signaling Technology). HRP labeled goat-anti-rabbit (sc-2054, 1:5,000, Santa Cruz Biotechnologies) was used as secondary antibody. For visualization, the SuperSignal West Pico PLUS kit (Fisher Scientific) was used and detection was conducted on a ChemiDoc System (Bio-Rad Laboratories, Vienna, Austria).

Gottschalk B., Koshenov Z., Bachkoenig O.A., Rost R., Malli R, & Graier W.F. (2022). MFN2 mediates ER-mitochondrial coupling during ER stress through specialized stable contact sites. Frontiers in Cell and Developmental Biology, 10, 918691.

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Protein Extraction and Analysis from Brain Tissues

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Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0°C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500μl, 5μl aprotinin, PMSF 50μl, Na 3 VO 4 100μl and NaF 20μl in 4.32 ml PBS. Extracts were clari ed by centrifugation at 4°C (13,200 g for 20 minutes). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE (43) . The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), a-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies.
Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.

Vanan S., Zeng X., Chia S.Y., Varnäs K., Zhang K., Jiang M., Padmanabhan P., Yu W., Zhou Z., Halldin C., Gulyás B., Tan E., & Li Z. (2020). Altered Striatal Dopamine Levels in Parkinson’s Disease VPS35 D620N Mutant Transgenic Aged Mice.

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