Rabbit anti tom20

Manufactured by Cell Signaling Technology

Sourced in United States

Rabbit anti-TOM20 is a primary antibody that specifically recognizes the TOM20 (Translocase of Outer Membrane 20) protein. TOM20 is a component of the mitochondrial outer membrane translocase complex and functions as a receptor for the import of nuclear-encoded mitochondrial proteins.

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Lab products found in correlation

Rabbit anti flag, by Cell Signaling Technology (2 mentions) Rabbit anti β actin, by Cell Signaling Technology (2 mentions) Prolongtm diamond antifade mountant, by Thermo Fisher Scientific (1 mentions) Dm5500b epifluorescence microscope, by Leica camera (1 mentions) Mitotrackertm red cmxros, by Thermo Fisher Scientific (1 mentions) Tcs sp8 confocal microscope, by Leica camera (1 mentions) Rabbit anti rps6, by Cell Signaling Technology (1 mentions) Mouse anti sirt1, by Cell Signaling Technology (1 mentions) Rabbit anti ampk, by Cell Signaling Technology (1 mentions) Rabbit anti p70s6k, by Cell Signaling Technology (1 mentions) Rabbit anti phospho ampk thr172 antibody, by Cell Signaling Technology (1 mentions) Ab 528428, by Santa Cruz Biotechnology (1 mentions) Mouse anti tom70, by Santa Cruz Biotechnology (1 mentions) Rabbit anti bip, by Cell Signaling Technology (1 mentions) Mouse anti strep mab, by IBA Lifesciences (1 mentions) Rabbit anti tom40, by Santa Cruz Biotechnology (1 mentions) Rabbit anti tom20, by Proteintech (1 mentions) Mouse anti β tubulin, by Merck Group (1 mentions) Mouse anti strep tag, by Qiagen (1 mentions) Mouse anti eea1, by BD (1 mentions)

7 protocols using rabbit anti tom20

Visualizing Mitochondrial Morphology in Cells

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Cells were grown at low density on coverslips. For visualization of mitochondrial morphology, cells were incubated with 200 nM MitoTracker ^TM Red CMXRos (Invitrogen, Eugene, OR) for 20 min. Cells were fixed in cold 4% paraformaldehyde and permeabilized and blocked with 0.3% Triton X-100 and 5% goat serum in PBS for 1 h at room temperature. Cells were incubated with the following primary antibodies diluted in 5% goat serum in PBS at 4 °C overnight: rabbit anti-TOM20 (1:300, Cell Signaling Technology, Danvers, MA, USA), chicken anti-MAP2 (1:1000, Invitrogen, Eugene, OR, USA), rabbit anti-Willin/FRMD6 (1:100, Cell Signaling Technology, Danvers, MA, USA). Corresponding fluorescent secondary antibodies (Alexa Fluor^TM 488, 568, 594, 647, 1:1000, Invitrogen, Eugene, OR, USA), were diluted in blocking buffer and incubated for 1 h at room temperature. Coverslips were mounted in ProLong^TM Diamond Antifade Mountant (Invitrogen, Eugene, OR, USA) and imaged with a Leica TCS SP8 confocal microscope using a 63× oil immersion objective or a Leica DM5500B epifluorescence microscope (Mannheim, Germany) using a 40× oil immersion objective.

Chen D., Yu W., Aitken L, & Gunn-Moore F. (2022). Willin/FRMD6 Mediates Mitochondrial Dysfunction Relevant to Neuronal Aβ Toxicity. Cells, 11(19), 3140.

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Western Blot Analysis of Muscle Proteins

Cited 1 time

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Protein extraction and western blot analysis were performed as reported from our previous publication [12 (link)]. The antibodies used were as follows: mouse anti-Pax7 (1 : 500, DSHB AB_528428), rabbit anti-MyoD (1 : 500, Santa Cruz sc-760), mouse anti-MyHC (1 : 500, DSHB MF20), rabbit anti-Tom20 (1 : 1000, Cell Signaling 42406), mouse anti-Sirt1 (1 : 1000, Cell Signaling 8469), rabbit anti-acH3 (1 : 1000, Cell Signaling 9649), rabbit anti-phospho AMPK (Thr172) antibody (1 : 1000, Cell Signaling 2535), rabbit anti-AMPK (1 : 1000, Cell Signaling 2603), rabbit anti-phospho P70S6K (Thr421/Ser424) (1 : 1000, Cell Signaling 9204), rabbit anti-P70S6K (1 : 1000, Cell Signaling 9202), rabbit anti-phospho RPS6 (Ser240/244) antibody (1 : 1000, Cell Signaling 2215), rabbit anti-RPS6 (1 : 1000, Cell Signaling 2217), and rabbit anti-tubulin antibody (1 : 500, Santa Cruz sc-9104). Densitometric analysis was performed using ImageQuant. Normalization of phosphorylated and total proteins was performed by using tubulin or vinculin. Finally, the ratio between the phosphorylated and total protein was indicated.

Pavlidou T., Marinkovic M., Rosina M., Fuoco C., Vumbaca S., Gargioli C., Castagnoli L, & Cesareni G. (2019). Metformin Delays Satellite Cell Activation and Maintains Quiescence. Stem Cells International, 2019, 5980465.

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Immunofluorescence Staining of SARS-CoV-2 Organelles

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Rabbit anti-beta-actin (Cell Signaling Technology #4967, RRID:AB_330288); mouse anti-beta tubulin (Sigma-Aldrich #T8328, RRID:AB_1844090); rabbit anti-BiP (Cell Signaling Technology #3177S, RRID:AB_2119845); mouse anti-EEA1 (BD Biosciences #610457, RRID:AB_397830, used at 1:200); mouse anti-ERGIC53 (Enzo Life Sciences #ALX-804-602-C100, RRID:AB_2051363, used at 1:200); anti-GM130; rabbit anti-GRP78 BiP (Abcam #Ab21685, RRID:AB_2119834); rabbit anti–SARS-CoV–nucleocapsid protein (NP) (Rockland #200-401-A50, RRID:AB_828403); rabbit anti-PDI (protein disulfide isomerase) (Cell Signaling Technology #3501, RRID:AB_2156433); mouse anti-Strep tag (QIAGEN #34850, RRID:AB_2810987, used at 1:5000); mouse anti-strepMAB (IBA Lifesciences #2-1507-001, used at 1:1000); rabbit anti–Strep-tag II (Abcam #ab232586); rabbit anti-Tom20 (Proteintech #11802-1-AP, RRID:AB_2207530, used at 1:1000); rabbit anti-Tom20 (Cell Signaling Technology #42406, RRID:AB_2687663); mouse anti-Tom22 (Santa Cruz Biotechnology #sc-101286, RRID:AB_1130526); rabbit anti-Tom40 (Santa Cruz Biotechnology #sc-11414, RRID:AB_793274); mouse anti-Tom70 (Santa Cruz #sc-390545, RRID:AB_2714192, used at 1:500); Rabbit anti-STX5 (Synaptic Systems 110 053, used at 1:500); and ActinStaining Kit 647-Phalloidin (Hypernol #8817-01, used at 1:400).

Gordon D.E., Hiatt J., Bouhaddou M., Rezelj V.V., Ulferts S., Braberg H., Jureka A.S., Obernier K., Guo J.Z., Batra J., Kaake R.M., Weckstein A.R., Owens T.W., Gupta M., Pourmal S., Titus E.W., Cakir M., Soucheray M., McGregor M., Cakir Z., Jang G., O’Meara M.J., Tummino T.A., Zhang Z., Foussard H., Rojc A., Zhou Y., Kuchenov D., Hüttenhain R., Xu J., Eckhardt M., Swaney D.L., Fabius J.M., Ummadi M., Tutuncuoglu B., Rathore U., Modak M., Haas P., Haas K.M., Naing Z.Z., Pulido E.H., Shi Y., Barrio-Hernandez I., Memon D., Petsalaki E., Dunham A., Marrero M.C., Burke D., Koh C., Vallet T., Silvas J.A., Azumaya C.M., Billesbølle C., Brilot A.F., Campbell M.G., Diallo A., Dickinson M.S., Diwanji D., Herrera N., Hoppe N., Kratochvil H.T., Liu Y., Merz G.E., Moritz M., Nguyen H.C., Nowotny C., Puchades C., Rizo A.N., Schulze-Gahmen U., Smith A.M., Sun M., Young I.D., Zhao J., Asarnow D., Biel J., Bowen A., Braxton J.R., Chen J., Chio C.M., Chio U.S., Deshpande I., Doan L., Faust B., Flores S., Jin M., Kim K., Lam V.L., Li F., Li J., Li Y.L., Li Y., Liu X., Lo M., Lopez K.E., Melo A.A., Moss FR I.I.I., Nguyen P., Paulino J., Pawar K.I., Peters J.K., Pospiech TH J.r., Safari M., Sangwan S., Schaefer K., Thomas P.V., Thwin A.C., Trenker R., Tse E., Tsui T.K., Wang F., Whitis N., Yu Z., Zhang K., Zhang Y., Zhou F., Saltzberg D., Hodder A.J., Shun-Shion A.S., Williams D.M., White K.M., Rosales R., Kehrer T., Miorin L., Moreno E., Patel A.H., Rihn S., Khalid M.M., Vallejo-Gracia A., Fozouni P., Simoneau C.R., Roth T.L., Wu D., Karim M.A., Ghoussaini M., Dunham I., Berardi F., Weigang S., Chazal M., Park J., Logue J., McGrath M., Weston S., Haupt R., Hastie C.J., Elliott M., Brown F., Burness K.A., Reid E., Dorward M., Johnson C., Wilkinson S.G., Geyer A., Giesel D.M., Baillie C., Raggett S., Leech H., Toth R., Goodman N., Keough K.C., Lind A.L., Klesh R.J., Hemphill K.R., Carlson-Stevermer J., Oki J., Holden K., Maures T., Pollard K.S., Sali A., Agard D.A., Cheng Y., Fraser J.S., Frost A., Jura N., Kortemme T., Manglik A., Southworth D.R., Stroud R.M., Alessi D.R., Davies P., Frieman M.B., Ideker T., Abate C., Jouvenet N., Kochs G., Shoichet B., Ott M., Palmarini M., Shokat K.M., García-Sastre A., Rassen J.A., Grosse R., Rosenberg O.S., Verba K.A., Basler C.F., Vignuzzi M., Peden A.A., Beltrao P, & Krogan N.J. (2020). Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms. Science (New York, N.y.), 370(6521), eabe9403.

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Western Blot Analysis of Hexokinase 2

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FLSs were plated 100,000 cells per well and were disrupted in lysis buffer after treatment and collection. Proteins were separated by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane through the semi-dry system (Invitrogen). After blocking for 1 h with 5% milk, blots were incubated overnight at 4°C with the following antibodies: rabbit anti-human/mouse HK2 at 1:500 (#2867, C64G5, Cell Signaling Technology, Danvers, MA), mouse anti-human HK2 at 1:500 (sc-374091, B8, Santa Cruz Co, Santa Clara, CA), mouse anti-human tubulin at 1:1,000 (Santa Cruz Co., Santa Clara, CA), rabbit anti-TOM20 at 1:500 (Cell Signaling Technology, Danvers, MA), and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and tubulin (Santa Cruz Co., Santa Clara, CA). Horseradish peroxidase–conjugated anti-mouse Immunoglobulin G (IgG) or anti-rabbit IgG (Cell Signaling Technology, Danvers, MA) was used as secondary antibody at 1:2,000 dilution. Membranes were developed using a chemiluminescence system (Clarity Western ECL, BIORAD, Hercules, CA). Images were captured by the ChemiDoc™ XRS+ System (BIORAD, Hercules, CA).

Torres A., Kang S., Mahony C.B., Cedeño M., Oliveira P.G., Fernandez-Bustamante M., Kemble S., Laragione T., Gulko P.S., Croft A.P., Sanchez-Lopez E., Miyamoto S, & Guma M. (2023). Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes. Frontiers in Immunology, 14, 1103231.

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Comprehensive Immunoblot Analysis of Mitochondrial Proteins

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For immunoblot analysis, the following antibodies were used: mouse anti-MFN1 (Abcam), rabbit anti-TOM20 (Cell Signaling Technology), mouse anti-TIM23 (BD Biosciences), rabbit anti-COXIV (Cell Signaling Technology), rabbit anti-UCHL1 (Cell Signaling Technology), mouse anti-tubulin (Developmental Studies Hybridoma Bank), rabbit anti–phospho-AMPKα (T172, Cell Signaling Technology), rabbit anti–phoshpo-ULK1 (S555, Cell Signaling Technology), mouse anti-AMPK (Abcam), rabbit anti-ULK1 (Cell Signaling Technology), rabbit anti-FUNDC1 (Novus Biologicals), mouse anti-VDAC1 (Abcam), mouse anti-NDUFS3 (Abcam), rabbit anti-Flag (Cell Signaling Technology), rabbit anti-HA (Cell Signaling Technology), mouse anti-Myc (MBL, Japan), mouse anti-TRIM63 (Santa Cruz Biotechnology), and rabbit anti-PKM antibody (Cell Signaling Technology). Peroxidase-conjugated secondary antibodies were purchased from the Jackson laboratory.

Ham S.J., Lee D., Xu W.J., Cho E., Choi S., Min S., Park S, & Chung J. (2021). Loss of UCHL1 rescues the defects related to Parkinson’s disease by suppressing glycolysis. Science Advances, 7(28), eabg4574.

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Analyzing Mitochondrial Protein TOM20 Levels

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The levels of mitochondrial protein TOM20 were analyzed by indirect immunofluorescence. Briefly, cells were grown and differentiated on glass coverslips and then fixed in 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, and incubated for 1 h with 1:100 rabbit anti-TOM20 (Cell Signaling, Danvers, MA, USA) in a buffer containing 50 mM Tris–HCl, pH 7.7; 0.1 M NaCl; and 2% bovine serum albumin. After antibody removal and several washes with the mentioned buffer, bound antibodies were detected by incubating the cells for 30 min with 1:100 AlexaFluor 488 anti-rabbit as the secondary antibody (Thermofisher Scientific, Waltham, MA USA). Images were captured using a confocal microscope TCS SP8 (Leica, Wetzlar, Germany) and analyzed using ImageJ software (NIH, Bethesda, MD, USA). Three independent experiments were quantified with four fields of view (areas) analyzed in 25–30 myotubes per condition per each separate experiment.

Abrigo J., Olguín H., Tacchi F., Orozco-Aguilar J., Valero-Breton M., Soto J., Castro-Sepúlveda M., Elorza A.A., Simon F, & Cabello-Verrugio C. (2023). Cholic and deoxycholic acids induce mitochondrial dysfunction, impaired biogenesis and autophagic flux in skeletal muscle cells. Biological Research, 56, 30.

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Immunoblotting Procedure for Cell Lysates

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Cells were lysed using 2X SDS-PAGE sample buffer (24 mM Tris-HCl, pH 6.8, 10% glycerol, 0.8% SDS, and 286 mM β-mercaptoethanol). Samples were subjected to SDS-PAGE on 4–20% Mini-PROTEAM^R TGX^™ gels (Bio-Rad, 4561096) and transferred to a nitrocellulose membrane (Bio-Rad, 1704159). The primary antibodies used in this study were: rabbit anti-ATF4 (Cell Signaling Technologeis, 11815s, 1:625), rabbit anti-β-actin (Cell Signaling Technologies, 4970S, 1:2500), rabbit anti-P-EIF2a (Cell Signaling Technologies, 3398S, 1:1000), mouse anti-EIF2α (Fisher Scientific, AHO0802, 1:1000), rabbit anti-β-Tubulin (Thermo Fisher, 32–2600, 1:2500), rabbit anti-UCP1 (Abcam, ab10983, 1:2500), rabbit anti-Flag (Cell Signaling Technologies, 2368S, 1:5000), rabbit anti-PKM2 (Cell Signaling Technologies, 4053S, 1:1000), rabbit anti-Tom20 (Cell Signaling Technologies, 42406S, 1:2500). Blots were incubated with antirabbit IgG (Cell Signaling Technologies, 7076S, 1:10000) or anti-mouse IgG (Cell Signaling Technologies, 7074S, 1:10000) and imaged using an analyticjena imaging system. Digital images were processed and quantified using ImageJ software.

Choe M, & Titov D.V. (2023). Genetically encoded tool for manipulation of ΔΨm identifies the latter as the driver of integrative stress response induced by ATP Synthase dysfunction. bioRxiv.

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