Fl 145

Manufactured by Santa Cruz Biotechnology

Sourced in United States

The FL-145 is a laboratory instrument used for fluorescence detection. It is designed to measure the intensity of fluorescent signals emitted from various samples.

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Lab products found in correlation

Retronectin, by Takara Bio (3 mentions) Stemspan, by STEMCELL (3 mentions) Mitotracker cmxros, by Thermo Fisher Scientific (3 mentions) Stem cell factor (scf), by Thermo Fisher Scientific (3 mentions) Lab tek 2 chamber slide, by Thermo Fisher Scientific (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) F1804, by Merck Group (2 mentions) Goat anti mouse alexa488 conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions) Fluoview fv10i, by Olympus (2 mentions) 2e3gc12fb2ae2, by Abcam (2 mentions) Chloracetamide, by Merck Group (2 mentions) Gfp trap agarose, by Proteintech (2 mentions) Ab6673, by Abcam (2 mentions) Mouse anti gapdh 6c5, by Thermo Fisher Scientific (2 mentions) N ethylmaleimide, by Merck Group (2 mentions) Mycoalert kit, by Lonza (2 mentions) Edta free protease inhibitor cocktail, by Roche (2 mentions) Phosphostop, by Roche (2 mentions) Lsm 710, by Zeiss (2 mentions) Lsm 780, by Zeiss (2 mentions)

24 protocols using fl 145

Subcellular Fractionation and Immunoblotting

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Subcellular fractionation was performed as described in Frezza et al.[38 (link)]. Mitochondrial and cytosolic fractions were obtained and were blotted with a mouse monoclonal antibody anti-TOM20 (1:5000) ref. FL-145 (Santa Cruz Biotechnology Santa Cruz, USA) and a goat polyclonal antibody anti-Actin (1:2000) ref sc1616 (Santa Cruz Biotechnology Santa Cruz, USA). Cells were lysed in RIPA buffer, in the presence of phosphatase and protease inhibitors. Similar amounts of total protein were resolved by SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare, Little Chalfont, England). We have used as primary antibodies, the ones already referred in the immunohistochemistry section (all at a dilution of 1:1000). For protein detection, a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) and a luminescence system (Perkin-Elmer, Foster City, USA) were used. Membranes were re-probed with a mouse monoclonal antibody anti-TOM20 (1:5000) ref. FL-145 (Santa Cruz Biotechnology Santa Cruz, USA) for control of the protein loading. Protein expression was quantified using the Bio-Rad Quantity One 1-D Analysis software.

Ferreira-da-Silva A., Valacca C., Rios E., Pópulo H., Soares P., Sobrinho-Simões M., Scorrano L., Máximo V, & Campello S. (2015). Mitochondrial Dynamics Protein Drp1 Is Overexpressed in Oncocytic Thyroid Tumors and Regulates Cancer Cell Migration. PLoS ONE, 10(3), e0122308.

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Antibody Characterization for Protein Profiling

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The following antibodies were used: CryAB rabbit polyclonal antibody (Millipore, Cat# ABN185, Darmstadt, Germany), 1:1 K for WB, 1:4 K for immunofluorescence (IF); Histone mouse monoclonal antibody (Fisher Scientific, Cat# AHO1432, Waltham, MA, 1:200 for WB); NFκB rabbit polyclonal antibody (C-20, Santa Cruz, Cat# sc-372, Santa Cruz, CA, 1:650 for WB, 1:750 for IF); Sox10 goat polyclonal antibody (N-20, Santa Cruz, Cat# sc-17342, 1:300 for IF); Alix mouse monoclonal antibody (3A9, Cell Signaling, Cat# 2171 T, 1:1 K for WB); GFAP chicken polyclonal antibody (Aves, 1:4 K for IF); Flotillin-1 rabbit polyclonal antibody (D2V7J, Cell Signaling, Cat# 18634, 1:1 K for WB); β-actin mouse monoclonal antibody (AC-74, Millipore, Cat# A2228, 1:1 K for WB); Tomm20 rabbit polyclonal antibody (FL-145, Santa Cruz, Cat# sc-11415, 1:1 K for WB); CD63 biotinylated antibody (Miltenyi Biotec, Cat# 130–108-922, Auburn, CA, 1:15 for IF); BLBP mouse monoclonal antibody (Abcam, Cat# ab131137, 1:2 K for IF) and p75-NGFR rabbit polyclonal antibody (Millipore, Cat# AB1554, 1:5 K). Recombinant chicken Anosmin1 (MyBioSource, Cat# MBS963562-COA, San Diego, CA) was used at 5 nM while recombinant CryAB protein (MyBioSource, Cat# MBS964495) and recombinant myoglobin (MyBioSource, Cat# MBS142891) were used at 50 ng/ml unless stated otherwise.

Saglam A., Calof A.L, & Wray S. (2020). Novel factor in olfactory ensheathing cell-astrocyte crosstalk: Anti-inflammatory protein α-crystallin B. Glia, 69(4), 1022-1036.

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Immunofluorescence and Immunoblotting of Irgm1

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A rabbit polyclonal antibody (3266 [5] (link)) and a mouse monoclonal antibody (1B2 [16] (link)) to Irgm1 have been described previously. In the studies described here, 3266 was used for immunoprecipitations and immunoblotting; 1B2 for immunofluorescence. Rabbit monoclonal antibody EP892Y (Abcam, Cambridge, MA) to the Golgi matrix protein GM130, and rabbit polyclonal antibody FL-145 (Santa Cruz Biotechnology, Dallas TX) to mitochondrial outer membrane protein TOM20 were also used for immunofluorescence.

Henry S.C., Schmidt E.A., Fessler M.B, & Taylor G.A. (2014). Palmitoylation of the Immunity Related GTPase, Irgm1: Impact on Membrane Localization and Ability to Promote Mitochondrial Fission. PLoS ONE, 9(4), e95021.

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Immunofluorescent Staining of Mitochondria

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Cells were cultured on poly-D-lysine coated glass slides and fixed with methanol and 4% formaldehyde solution. If Mitotracker CMXRos (M-7512, Thermo Fisher Scientific) was used, it was added before fixation according to the manufacturer’s instructions. Fixed cells were washed and blocked for 1 h with 1% goat serum in PBS, and stained with primary antibodies rabbit anti-Tom20 (1:500, FL-145, Santa Cruz Biotechnology) and mouse anti-FLAG (1:200, F1804, Sigma-Aldrich) in a humidified chamber at 4 °C overnight. Cells were washed again and stained with secondary antibodies goat anti-rabbit Cy5 (1:200, ab97077, Abcam), and goat anti-mouse Alexa Fluor 568 (1:500, ab175701, Abcam) for 1.5 h. After additional washing steps and staining with DAPI, cells were imaged using a Zeiss LSM 780 confocal microscope.

Schmiderer L., Yudovich D., Oburoglu L., Hjort M, & Larsson J. (2022). Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import. Scientific Reports, 12, 18687.

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Comprehensive Protein Detection Protocol

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The following antibodies were used for Western blot and immunofluorescense assays: rabbit anti-Beclin 1 (PD017; Medical & Biological Laboratories ;1:3000) guinea pig anti-K5 (GP-CK5; Progen Biotechnik; 1:5000 for western blot, 1:100 for immunofluorescence), mouse anti-K10 (MAB3230; Millipore; WB: 1:5000, IF: 1:200), mouse anti-K14 (ab7800; Abcam; WB: 1:20,0000, IF: 1:500), rabbit anti-K1 (905204; BioLegend; 1:5000), rabbit anti-loricrin (ab24722; Abcam; 1:2000), rabbit anti-filaggrin (905804; BioLegend; WB: 1:10,000, IF: 1:200), rabbit anti-survivin (#2808; Cell Signaling Technology; 1:100), rat anti-integrin α6 (555734; BD Pharmingen; 1:200), rabbit anti-integrin α6 (#3750; Cell Signaling Technology; 1:1000), anti-integrin β4 (553745; BD Pharmingen; 1:100), rabbit anti-Tom20 (FL-145; Santa Cruz Biotechnology; 1:500), mouse anti-GM130 (610823; BD Pharmingen; 1:100), rat anti-transferrin receptor (553266; BD Pharmingen; 1:100), rabbit anti-EEA1 (#3285 S, Cell Signaling Technology; 1:100), rabbit anti-Rab11 (#5589 S; Cell Signaling Technology; 1:100), mouse anti-γ-tubulin (T6557; Sigma-Aldrich; 1:100), rabbit anti-p63 (H-137; Santa Cruz Biotechnology; 1:50), and mouse anti-actin (#MAB-1501; Millipore; 1:10,000). All other chemicals were purchased from Nacalai Tesque.

Noguchi S., Honda S., Saitoh T., Matsumura H., Nishimura E., Akira S, & Shimizu S. (2019). Beclin 1 regulates recycling endosome and is required for skin development in mice. Communications Biology, 2, 37.

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Mitochondrial Morphology Characterization

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Cultured cells were seeded to 18 mm² glass coverslips. Coverslips were incubated with MitoTracker CMXRos (Invitrogen Molecular Probes, Carlsbad, CA, USA), followed by fixation in 4% paraformaldehyde in PBS. For immunolabeling of the translocase of the outer mitochondrial membrane-20 protein (TOM20), cells were permeabilized with 0.1% TX-100 in PBS, followed by blocking in 10% normal goat serum and incubation with anti-TOM20 monoclonal antibody FL-145 at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Coverslips were incubated with goat anti-mouse Alexa488-conjugated secondary antibody at 1:100 dilution (Invitrogen Molecular Probes, Carlsbad, CA, USA), followed by staining with diaminophenylindole (DAPI) and mounting in 50% glycerol in PBS. Coverslips were imaged on an Olympus Fluoview FV-10i (Olympus, Center Valley, PA, USA) with a 60× UPLSAP60xW objective with aperture 1.2 and 3× optical zoom at room temperature. For scoring of mitochondrial morphology, individual cell profiles on confocal micrographs were scored as predominantly reticular if they had fewer than three instances of fragmented mitochondria. Cells were scored as predominantly fragmented if they displayed fewer than three instances of mitochondrial interconnection. All others were scored as having intermediate mitochondrial morphology.

Jones E., Gaytan N., Garcia I., Herrera A., Ramos M., Agarwala D., Rana M., Innis-Whitehouse W., Schuenzel E, & Gilkerson R. (2016). A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1. Cellular and Molecular Life Sciences, 74(7), 1347-1363.

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Multiplexed Immunostaining of Mitochondrial Proteins

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Sorted cells were resuspended in 50 μl of StemSPAN (StemCell Technologies) supplemented with 50 ng/ml SCF (Peprotech) and 50 ng/ml TPO (Peprotech) then seeded on Lab-Tek™ II Chamber Slide (Thermo Fisher Scientific) coated with Retronectin (Clonetech). Samples were then immunostained as described previously (Bonora et al., 2018 (link)). Rabbit anti-Ki67 (D3B5, CST, dilution 1:100), rabbit polyclonal anti-TOM20 (FL-145, Santa Cruz, dilution 1:100), mouse monoclonal anti-ATP5A1 (15H4C4, Invitrogen, dilution 1:100), rabbit polyclonal anti-NDUFV1 (Novusbio, dilution 1:100) and mouse monoclonal anti-SDHA (2E3GC12FB2AE2, abcam, dilution 1:100) were used for the detection. Z-stack were acquired on a Leica SP5 equipped with 63× oil immersion lens (NA 1.4), pinhole set at 1 airy unit with voxel size 80/200 nm on XY/Z. Stacks were deconvolved using the Richardson-Lucy algorithm (Sage et al., 2017 (link)) using measured PSF. Representative image renderings were obtained by Imaris 7 (Bitplane).

Morganti C., Bonora M., Ito K, & Ito K. (2019). Electron transport chain complex II sustains high mitochondrial membrane potential in hematopoietic stem and progenitor cells. Stem cell research, 40, 101573.

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Immunofluorescence Labeling of MAP6 Proteins

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A primary antibody directed against a central repeat motif of MAP6 proteins (antibody 23N), described previously [19 (link)], was used to label all the MAP6 isoforms. The antibody against the alpha-1 subunit of dihydropyridine receptor (DHPR) was from Abcam (#Ab2862), the antibodies against β-tubulin (TUB2.1, #T5201) and alpha-actinin (A-7811) were from Sigma, and the antibody against Golgi apparatus was from Santa Cruz (FL-145). Antibodies against RyR1, triadin T95, tyrosinated tubulin (YL1/2), and detyrosinated tubulin were previously described [35 (link)–37 (link)]. The antibody against SERCA was kindly provided by Dr. M.-J. Moutin [38 (link)].

Sébastien M., Giannesini B., Aubin P., Brocard J., Chivet M., Pietrangelo L., Boncompagni S., Bosc C., Brocard J., Rendu J., Gory-Fauré S., Andrieux A., Fourest-Lieuvin A., Fauré J, & Marty I. (2018). Deletion of the microtubule-associated protein 6 (MAP6) results in skeletal muscle dysfunction. Skeletal Muscle, 8, 30.

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Mitochondrial Structure Visualization and Quantification

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Mitochondria were stained using immunocytochemistry (ICC) (as described above) with primary antibodies against TOM20 (FL145 Santa Cruz Biotech, Dallas, Texas; 1:100); and ATPB (AB5452 Abcam, Cambridge, UK; 1:500). Imaging was performed by confocal microscopy (Leica TCS S5 microscope, Leica microsystems, Wetzlar, Germany). Image processing, three-dimensional (3D)-modeling and quantitative analysis of mitochondrial structures were performed using the Image-Pro Plus software (version 7.0) (Media Cybernetics), as described previously [32 (link), 36 (link)]. Further details are provided in Additional file 1: Supplemental methods.

Røsland G.V., Dyrstad S.E., Tusubira D., Helwa R., Tan T.Z., Lotsberg M.L., Pettersen I.K., Berg A., Kindt C., Hoel F., Jacobsen K., Arason A.J., Engelsen A.S., Ditzel H.J., Lønning P.E., Krakstad C., Thiery J.P., Lorens J.B., Knappskog S, & Tronstad K.J. (2019). Epithelial to mesenchymal transition (EMT) is associated with attenuation of succinate dehydrogenase (SDH) in breast cancer through reduced expression of SDHC. Cancer & Metabolism, 7, 6.

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Lung Tumor Lysate Preparation and Analysis

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Whole cell lysates from lung tumors isolated from mice were prepared as previously described^{7 (link)}. Briefly, tumors were homogenized in buffer containing phosphatase and protease inhibitors (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM pyrophosphate, 1 mM sodium orthovanadate, protease inhibitor tablet), centrifuged and supernatant was normalized, aliquoted and stored in −80°C freezer. Lysates were separated on 4–12% Bis-Tris protein gels (Thermo), transferred to PVDF membrane and probed with the following antibodies: SP-C (1:5000, AB3786 Milipore); Glut1 (1:2000, GT11-A, Alpha Diagnostic); Ndufs1 (1:2000, ab169540, abcam); Ndufs1 (1:2000, sc-271510, Santa Cruz); Ndusv1 (1:500, 11283–1-AP, Proteintech), Ndufv2 (1:2000, sc-271620, Santa Cruz), Tom20 (1:10000, FL-145, Santa Cruz), Tom40 (1:2000, 18409–1-AP, Proteintech); Tom70 (1:2000, 14528–1-AP, Proteintech); Tim23 (1:2000, 11123–1-AP, Proteintech); actin (1:8000, #4970 and #3700, Cell Signaling Technology). Intensity of bands was quantified using Image J.

Momcilovic M., Jones A., Bailey S.T., Waldmann C.M., Li R., Lee J.T., Abdelhady G., Gomez A., Holloway T., Schmid E., Stout D., Fishbein M.C., Stiles L., Dabir D.V., Dubinett S.M., Christofk H., Shirihai O., Koehler C.M., Sadeghi S, & Shackelford D.B. (2019). In vivo imaging of mitochondrial membrane potential in non-small cell lung cancer. Nature, 575(7782), 380-384.

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