Anti tomm20

Manufactured by Abcam

Sourced in United States

Anti-Tomm20 is a primary antibody that targets the Translocase of Outer Mitochondrial Membrane 20 (TOMM20) protein. TOMM20 is a key component of the protein import machinery responsible for the translocation of nuclear-encoded proteins into the mitochondria.

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Lab products found in correlation

Anti ha, by Abcam (3 mentions) Anti tomm20 f 10, by Santa Cruz Biotechnology (2 mentions) Anti hsp60 lk1, by Santa Cruz Biotechnology (2 mentions) Anti filamin 1 e 3, by Santa Cruz Biotechnology (2 mentions) Anti arp3, by Proteintech (2 mentions) Anti cofilin e 8, by Santa Cruz Biotechnology (2 mentions) Anti gelsolin gs 2c4, by Abcam (2 mentions) Anti adf, by Abcam (2 mentions) Anti cortactin 4f11, by Merck Group (2 mentions) Anti pericentrin, by BioLegend (2 mentions) Anti wave1, by Santa Cruz Biotechnology (2 mentions) Anti whamm, by Abcam (2 mentions) Anti myosin19, by Abcam (2 mentions) Anti myova, by Santa Cruz Biotechnology (2 mentions) Alexa fluor 647 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594 goat anti mouse, by Thermo Fisher Scientific (2 mentions) Irdye 800cw donkey anti rabbit, by LI COR (2 mentions) Irdye 800cw donkey anti mouse, by LI COR (2 mentions) Alexa fluor 647, by Thermo Fisher Scientific (2 mentions)

19 protocols using anti tomm20

Antibody Staining for Cytoskeletal Proteins

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1° antibodies: anti-alpha-tubulin clone DM1A (Sigma, T6199), anti-Tomm20 (Abcam, ab78547), anti-Tomm20 F-10 (SantaCruz, sc-17764), anti-HSP60 LK1 (SantaCruz, sc-59567), anti-Filamin 1 E-3 (SantaCruz, sc17749), Anti-Arp3 (Proteintech, 13822-1-AP), anti-Cofilin E-8 (SantaCruz, sc-376476), Anti-Gelsolin GS-2C4 (Abcam, ab11081), anti-ADF (Abcam, ab186754), anti-Cortactin 4F11 (Millipore, 05–180), Anti-pericentrin (Biolegend, 923701), Anti-INF2-CAAX and pan-INF2 (gifts from H.Higgs), Anti-N-WASP (CST, 4848T), Anti-VASP (CST, 3112), anti-WASHC1 (Atlas, HPA002689), Anti-WAVE1 (SantaCruz, sc-271507), Anti-WHAMM (Abcam, ab122572), Anti-Myosin19 (Abcam, ab174286), Anti-MyoVa (SantaCruz, sc-365986). 2° antibodies: Goat-anti-rabbit Alexa Fluor 555 (Invitrogen, A-21429), donkey-anti-rabbit AlexaFluor 594 (Invitrogen, R37119), Donkey-anti-Rabbit Alexa Fluor 647 (Invitrogen, R37119), Goat anti-Mouse Alexa Fluor 488 (Invitrogen, A-11001), Goat-anti-mouse Alexa Fluor 594 (Invitrogen, A11032), Goat-anti-mouse, Alexa Fluor 647 (Invitrogen, A32728), IRDye 800CW Donkey anti-rabbit (Licor, 926–32213), and IRDye 800CW Donkey anti-mouse (Licor, 926–32212).

Moore A.S., Coscia S.M., Simpson C.L., Ortega F.E., Wait E.C., Heddleston J.M., Nirschl J.J., Obara C.J., Guedes-Dias P., Boecker C.A., Chew T.L., Theriot J.A., Lippincott-Schwartz J, & Holzbaur E.L. (2021). Actin cables and comet tails organize mitochondrial networks in mitosis. Nature, 591(7851), 659-664.

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Purification and Analysis of SCF Complexes

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The SCF components such as human influenza hemagglutinin (HA)-Skp1, Cul1, Myc-Rbx1, and FLAG-Fbxo7 or FLAG-Fbxo7(ΔF-box) were transfected in HEK293T cells by using polyethylenimine. After 48 h of transfection, the cells were harvested and resuspended in lysis buffer (LB) (25 mM Tris–HCl, pH 7.5, 225 mM KCl and 1% NP-40) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and phosphatase inhibitors (10 mM NaF and 1 mM Na₃VO₄). The lysates were incubated with agarose anti-FLAG M2 beads (Sigma-Aldrich, St. Louis, MO) for 6 h at 4°C with rocking. Beads were washed with LB and the SCF complexes eluted with FLAG elution buffer (300 µg/ml of peptide FLAG in 10 mM HEPES pH 7.9, 225 mM KCl, 1.5 mM MgCl₂, and 0.1% NP-40) for 1 h at 4°C with rocking. The eluates were stored in 15% glycerol at −20°C until use. To evaluate the purification of SCF complexes, immunoblotting was performed and probed using anti-Fbxo7 (ABN1038, Merck Millipore, Watford, UK), anti-HA (Abcam, Cambridge, UK), anti-Gsk3β (Santa Cruz Biotechnologies, CA, USA), anti-Tomm20 (Abcam, Cambridge, UK), or anti-myc (Cell Signaling Technologies, MA, USA). The concentration of the complexes was determined against known concentrations of BSA by Coomassie blue staining of the gel. The densitometry of the bands was determined by ImageJ.

Teixeira F.R., Randle S.J., Patel S.P., Mevissen T.E., Zenkeviciute G., Koide T., Komander D, & Laman H. (2016). Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease. Biochemical Journal, 473(20), 3563-3580.

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Antibody Staining for Cytoskeletal Proteins

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Paclitaxel Effects on Axonal Mitochondria

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E15 DRG neurons grown in microfluidic cultures were treated with 60 nM paclitaxel to the axonal compartment for 48 hours, and fixed at room temperature with 4% PFA diluted 1:2 in media for 10 min, then undiluted 4% PFA for an additional 10 min. Cultures were permeabilized and blocked as above and incubated overnight at 4°C with the following antibodies: anti-Tomm20 (1:300; Abcam), and anti-Tuj1. Cultures were then stained with AlexaFluor antibodies and counterstained with DAPI as above. Images were acquired throughout the axon compartment using a 60× 1.4NA oil objective, and Tomm20 mitochondrial length was measured manually using NIH ImageJ by an experimenter blind to condition. 1078 (vehicle) and 1049 (paclitaxel) axonal mitochondria were measured across three independent experiments.

Pease-Raissi S.E., Pazyra-Murphy M.F., Li Y., Wachter F., Fukuda Y., Fenstermacher S.J., Barclay L.A., Bird G.H., Walensky L.D, & Segal R.A. (2017). Paclitaxel reduces axonal Bclw to initiate IP3R1-dependent axon degeneration. Neuron, 96(2), 373-386.e6.

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Molecular Mechanisms of Anti-Inflammatory Agents

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3-Amino-1, 2, 4-triazole (#A8056), N-acetyl-l-cysteine (#A7250) and Chloroquine (#C6628), were purchased from Sigma-Aldrich. Recombinant mouse TNF-α (#MTA00B) and IL-6 (#M6000B) enzyme-linked immunosorbent assay (ELISA) kits (Quantikine) for cytokine analysis were bought from R&D Systems (Minneapolis, MN). Anti-PMP70 (#sab4200181, Sigma-Aldrich), anti-Catalase (#ab16731, Abcam), anti-SQSTM1/p62 (#H00008878-M01, Abnova), anti-LC3 (#L8918, Sigma-Aldrich), anti-Pex5 (#GTX109798, GeneTex), anti-Pex7 (#20614-1-AP, Proteintech), anti-DBP (#TA308904, OriGene), anti-ACOX1 (#10957-1-AP, Proteintech), anti-UBXD8 (#NB100-1296, Novs), anti-HA (#ab130275, Abcam), anti-Pex1 (#13669-1-AP, Proteintech), anti-Pex16 (#14186-1-AP, Proteintech), nti-Pex3 (#247042, Abcam), anti-Pex9 (PA5-22129, Invitrogen), anti-Tomm20 (#ab56783, Abcam), anti-NF-κB (#sc-8008, Santa Cruz), anti-IκBα (#9242S, CST), anti-p-IκBα (#2859S, CST), anti-4-HNE (#ab46545, Abcam) and anti-β-actin (#sc-47778, Santa Cruz).

Mu Y., Maharjan Y., Kumar Dutta R., Wei X., Kim J.H., Son J., Park C, & Park R. (2021). Pharmacological inhibition of catalase induces peroxisome leakage and suppression of LPS induced inflammatory response in Raw 264.7 cell. PLoS ONE, 16(2), e0245799.

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Fluorescent Imaging of Organelle Distribution

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We used the following antibodies: anti-TOMM20 (Abcam, USA, cat. ab56783); anti-GM-130 (Abcam, USA, cat. ab52649); anti KDEL (Abcam, USA, cat. ab12223). Control and treated macrophages seeded on chamber slides (0.4 × 10⁶/slide) were fixed in 4% formaldehyde in PBS with 0.05% Triton X-100. After washing in PBS–Tween 20, fixed cells were blocked in casein blocking buffer (Bio-Rad, USA) with 0.05% Tween 20 and subsequently incubated in blocking buffer with a 1:200 dilution of primary antibodies and rhodamine–phalloidin overnight at 4 °C. Subsequently, after extensive washing in PBS–Tween 20, cells were mounted in ProLong^® Diamond Antifade with DAPI and observed under a Nikon fluorescence microscope. All experiments were performed in triplicate. The difference in organelle distribution between untreated and treated cells was calculated using the fluorescent area (normalized to total cell area using actin fluorescence) and corrected total cell fluorescence of individual cells using the image-processing ImageJ program.

Chanana P., Uosef A., Vaughn N., Suarez-Villagran M., Ghobrial R.M., Kloc M, & Wosik J. (2022). The Effect of Magnetic Field Gradient and Gadolinium-Based MRI Contrast Agent Dotarem on Mouse Macrophages. Cells, 11(5), 757.

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Immunostaining of Neuronal Markers

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Slice cultures were fixed with 4% paraformaldehyde overnight at 4 °C and incubated in blocking buffer (10% normal goat serum in 0.1% Triton-X in PBS) for 1 h. Cultures were left in primary antibody (1:200 anti-tau, abcam; cat. #ab75714), 1:500 anti-MAP2 (BD Pharmingen, San Diego, CA, USA; cat. #556320), 1:200 anti-DRP1 EPR19274 (abcam; cat. #ab184247), 1:200 anti-NCX1 EPR12739 (abcam; cat. #ab177952), and 1:200 anti-TOMM20 (abcam; cat. #ab56783) overnight at 4 °C, washed 3× with PBS, and incubated with respective secondary antibody conjugated to Alexa Fluor 488 or 647 (1:500; ThermoFisherScientific). Fluorescence micrographs were taken under a ×20 objective using the EVOS FL Microscope (ThermoFisherScientific). The following filter cubes were used for this study: EVOS™ Light Cube, GFP cat. AMEP4651; EVOS™ Light Cube, RFP cat. AMEP4652; EVOS™ Light Cube, Cy™5 cat. AMEP4656. The Look Up Table (LUT) applied to display CellROX Green fluorescence intensity is linear and found in ImageJ (16-color LUT). Colocalization of TOMM20 and Drp1 immunostaining was determined using the Coloc 2 plugin (https://imagej.net/Coloc_2) in ImageJ.

Omelchenko A., Shrirao A.B., Bhattiprolu A.K., Zahn J.D., Schloss R.S., Dickson S., Meaney D.F., Boustany N.N., Yarmush M.L, & Firestein B.L. (2019). Dynamin and reverse-mode sodium calcium exchanger blockade confers neuroprotection from diffuse axonal injury. Cell Death & Disease, 10(10), 727.

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Intracellular VARS Protein Immunofluorescence

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For immunofluorescence (IF) staining of intracellular VARS protein, 50,000 fibroblasts of patients 4, 5 and a control individual were seeded on 12 mm diameter Coverslips and 24 h later fixed with 4% paraformaldehyde (PFA) for 20 min at RT. Fibroblasts were permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 2 min, blocked with 0.5% bovine serum albumin and 0.2% goat serum for 1 h and incubated overnight at 4 °C with following primary antibodies: anti-VARS protein (1:500; Atlas Antibodies, HPA046710), anti-KDEL (1:100; Enzo Life Sciences, 10C3), anti-Golgin-97 (1:200, Life Technologies, A21270), and anti-TOMM20 (1:200, Abcam, ab56783). With intermediate PBS washing steps, the secondary antibodies goat anti-rabbit IgG (Alexa Fluor 488) and goat anti-mouse IgG (Alexa Fluor 594) (both 1:500; Life Technologies) were added for 1 h at RT. Nuclei were stained for 10 min with Hoechst 33342 (1:10,000, Life Technologies). Coverslips were mounted (Dako) and images were taken with a Zeiss LSM700 confocal microscope using a ×63/1.40 plan-apochromatic objective. Possible cross-talk of the fluorescence channels was excluded by using frame-by-frame scanning.

Siekierska A., Stamberger H., Deconinck T., Oprescu S.N., Partoens M., Zhang Y., Sourbron J., Adriaenssens E., Mullen P., Wiencek P., Hardies K., Lee J.S., Giong H.K., Distelmaier F., Elpeleg O., Helbig K.L., Hersh J., Isikay S., Jordan E., Karaca E., Kecskes A., Lupski J.R., Kovacs-Nagy R., May P., Narayanan V., Pendziwiat M., Ramsey K., Rangasamy S., Shinde D.N., Spiegel R., Timmerman V., von Spiczak S., Helbig I., Weckhuysen S., Francklyn C., Antonellis A., de Witte P, & De Jonghe P. (2019). Biallelic VARS variants cause developmental encephalopathy with microcephaly that is recapitulated in vars knockout zebrafish. Nature Communications, 10, 708.

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Immunofluorescence Staining of Fixed Cells

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PFA fixed cell cultures (4% PFA except for CPT1a staining where 2% PFA was used), spheroids (4% PFA) or whole mount retinas (4% PFA) were subjected to immunofluorescence staining using the following isolectin conjugates or primary antibodies: isolectin GS-IB4-Alexa 488, isolectin GS-IB4-Alexa 568, isolectin GS-IB4-Alexa 647 (Molecular Probes), anti-CPT1a (Cell Signaling), anti-NG2 Chondroitin Sulfate Proteoglycan (Chemicon), anti-Tomm20 (Abcam) and anti-collagen IV (Southern Biotech). Alexa-488, -568 or -633 conjugated secondary antibodies were used (Molecular Probes). EdU, EU and HPG staining was performed using a Click-IT assay with Alexa fluor 555 according to manufacturer’s instructions.

Schoors S., Bruning U., Missiaen R., Queiroz K.C., Borgers G., Elia I., Zecchin A., Cantelmo A.R., Christen S., Goveia J., Heggermont W., Goddé L., Vinckier S., Van Veldhoven P.P., Eelen G., Schoonjans L., Gerhardt H., Dewerchin M., Baes M., De Bock K., Ghesquière B., Lunt S.Y., Fendt S.M, & Carmeliet P. (2015). Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature, 520(7546), 192-197.

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Molecular Mechanisms of Mitophagy in HK-2 Cells

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HK‐2 cells (Human, kidney proximal tubular 2) were obtained from ATCC (ATCC^® CRL‐2190™). The primary antibodies used in this study include anti‐PARKIN (Cat no: #2132, 1:500; Cell Signaling Technology), anti‐p‐PARKIN (Cat no: orb312554, 1:250, Biorbyt), anti‐PINK1 (Cat no: #6946, 1:500; Cell Signaling Technology), anti‐BNIP3L (Cat no: GTX111876, 1:1000, GeneTex), anti‐BNIP3 (Cat no: GTX10433, 1:1000, GeneTex), anti‐HuR (Cat no: #12582, 1:1000, Cell Signaling Technology), anti‐TOMM20 (Cat no: ab56783, 1:200, Abcam), anti‐LC3 (Cat no: NB100‐2220, 1:200, Novus Biologicals), anti‐COXIV (Cat no: 4850, 1:1000, Cell Signaling Technology), anti‐LAMP2 (Cat no: 49067, 1:200, Cell Signaling Technology), and anti‐β‐ACTIN (Cat no: A1978, 1:1000, Sigma‐Aldrich). Secondary antibodies‐rabbit/mouse IgG HRP, Alexa Fluor 546 anti‐mouse IgG (H + L), Alexa Fluor 488 anti‐goat IgG (H + L), Alexa Fluor 647 anti‐rabbit IgG (H + L) were purchased from Invitrogen™. 2′,7′‐dichlorofluorescin‐diacetate (DCFH2‐DA), JC‐1 and DAPI (4′,6‐diamidino‐2‐phenylindole) mount were purchased from Sigma Aldrich. Magna RIP™ RNA‐Binding Protein Immunoprecipitation Kit was procured from Sigma‐Aldrich. The SuperSignal West Femto Maximum Sensitivity Substrate detection reagents were purchased from Thermo Scientific Inc.

Yu S., Palanisamy K., Sun K., Li X., Wang Y., Lin F., Chen K., Wang I., Yu T, & Li C. (2021). Human antigen R regulates hypoxia‐induced mitophagy in renal tubular cells through PARKIN/BNIP3L expressions. Journal of Cellular and Molecular Medicine, 25(5), 2691-2702.

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