Commercially available antibodies were used to detect FK2 (Enzo Life Sciences, BML-PW8810), DDK tag (Origene, TA50011), HCV NS3 (TORDJI-22, Abcam), HCV core (ThermoFisher Scientific, MA1-080), HAV capsid (Commonwealth Serum Laboratories, K24F2), Dengue capsid (Dr. Tom Hobman, University of Alberta), STAT1 (Cell Signaling Technologies, CST9175), STAT2 (Santa Cruz, sc-476), Y701-phosphoSTAT1 (Cell Signaling Technologies, 9167S), Y690-phosphoSTAT2 (Cell Signaling Technologies, 88410S), PDLIM2 (Abcam, 246868), p65 NF-κB (Santa Cruz, sc-372), Actin (EMD Millipore, MAB1501), PARP-1 (BD Pharmingen, BD556362), Lamin (Zymed, 33–2000), B-tubulin (Abcam, AB6046), Anti-NS5a (gift from Charlie Rice, 9E10). For western blotting, goat anti-mouse IR Dye 800 (Licor, 926–32210) and goat anti-rabbit IR Dye 680 (Licor, 926–32221) were used. For immunofluorescence, Alexa Fluor 546 goat anti-mouse IgG (ThermoFisher Scientific, A11030), Alexa Fluor 488 goat anti-rabbit IgG, (ThermoFisher Scientific, A11008), or Alexa Fluor 647 goat anti-mouse IgG (ThermoFisher Scientific, A21236) were used.
Goat anti mouse irdye 800
Manufactured by LI COR
Sourced in United States, Germany
The Goat anti-mouse IRDye 800 is a secondary antibody conjugated with the IRDye 800 fluorescent dye. It is designed for use in immunoassays and other fluorescence-based applications that require the detection of mouse primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit irdye 680, by LI COR (17 mentions)
Goat anti rabbit irdye 800, by LI COR (9 mentions)
Goat anti mouse irdye 680, by LI COR (8 mentions)
Odyssey infrared imaging system, by LI COR (7 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Odyssey infrared scanner, by LI COR (2 mentions)
Goat anti rabbit hrp, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Hcv core, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Abcam (1 mentions)
Ddk tag, by OriGene (1 mentions)
Stat2, by Santa Cruz Biotechnology (1 mentions)
Pdlim2, by Abcam (1 mentions)
Parp 1, by BD (1 mentions)
Stat1, by Cell Signaling Technology (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
34 protocols using goat anti mouse irdye 800
1
Antibody Characterization for Protein Detection
2
Protein Analysis of RPE-1 Cells
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Retinal pigment epithelial‐1 cells used for protein analysis were plated at low density onto 6‐well cell plates (Eppendorf 30720113) in culture medium and treated for each experimental condition at 50% confluence. For whole‐cell protein analysis, cells were lysed with ice‐cold RIPA buffer containing protease and phosphatase inhibitors. Lysates were separated on a gradient gel (TGX, Bio‐Rad) and transferred to a PVDF membrane. Membranes were blocked with blocking buffer (LI‐COR Odyssey Blocking Buffer 927–40000) for 1 hr before probing with primary antibodies for p16 (Abcam ab108349), SQSTM1/p62 (Abcam ab56416), LC3B (Cell Signaling E5Q2K), and beta‐actin (Cell Signaling 8H10D10) in blocking buffer overnight at 4°C. Membranes were washed and probed with secondary antibodies (LI‐COR goat anti‐mouse IRDye800 and goat anti‐rabbit IRDye680) for 1 hr at room temperature and visualized using the LI‐COR Odyssey CLx Imaging System. Proteins were normalized to actin and quantified using ImageJ. Quantified results were tested for statistical significance in MATLAB using two‐way ANOVA and Bonferroni correction for multiple comparison analysis.
3
Investigating Phosphatase Regulation of Alpha-Synuclein
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Both protease and phosphatase inhibitors were purchased from Roche (Basel, Switzerland). Primary antibodies used in western blotting are as follows: Mouse anti-β-actin (1:5000, Proteintech), Rabbit anti-p-α-syn (1:1000, Abcam), Mouse anti-p-α-syn (1:1000, Wako), Mouse anti-α-syn (1:1000, Santa Cruz), Rabbit anti-Human α-syn (1:2000, Abcam), Mouse anti-demethylate-PP2A (1:1000, Millipore), Mouse anti-PP2A (1:1000, BD biosciences), Mouse anti-β-tubulin (1:1000, Abcam), Mouse anti-PPP2R2A (1:1000, CST), Mouse anti-PPP2R2B (1:1000, Abcam), Rabbit anti-PPP2R2D (1:1000, Abcam), Mouse anti-PPP2R5A (1:500, Santa Cruz), Rabbit anti-PPP2R5D (1:5000, Abcam), Rabbit anti-PPP2R5E (1:1000, Abcam), Mouse anti-LCMT-1 (1:1000, Abcam), Rabbit anti-PME-1 (1:1000, Millipore), Mouse anti-GAPDH (1:3000, Sigma), Mouse anti-c-Myc (1:5000, Clontech), and Mouse anti-FLAG (1:500, Santa Cruz). Secondary antibodies are as follows: Goat anti-Rabbit IRdye680 (LI-COR Bioscience), Goat anti-Mouse IRdye680 (LI-COR Bioscience), Goat anti-Rabbit IRdye800 (LI-COR Bioscience), Goat anti-Mouse IRdye800 (LI-COR Bioscience), Goat anti-Rabbit Alexa Fluor 488 (Thermo Fisher Scientific). The inhibitor of protein phosphatase methylesterase-1 (PME-1), AMZ30, was purchased from MedChem Express (Shanghai, China). Dulbecco’s Modified Eagle’s Medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco.
4
Protein expression analysis in cell extracts
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Cells were harvested and homogenized in RIPA buffer (Sigma-Aldrich® GmbH, St. Louis, MO, USA) containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.1% (v/v) sodium dodecyl sulfate and protease, phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentrations were determined by DC™ Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and equal amounts of proteins (25 µg) were separated by 8% and 10% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated with antibodies specific to Myosin (1:200), MuRF-1 (1:200), and Foxo (1:200) (all from Santa Cruz Biotechnology, Dallas, TX, USA), and GAPDH (1:10,000, Acris Antibodies GmbH, Herford, Germany), respectively, and appropriate secondary dye-conjugated antibodies goat anti-mouse IRdye800, IRdye650 (1:10,000; LI-COR®, Lincoln, NE, USA) to reveal protein bands for 1 h at room temperature. Membranes were scanned using the Odyssey SA Imaging System (LI-COR®, Lincoln, NE, USA). Experiments were performed in duplicates and the signal intensity was normalized to GAPDH.
5
Western Blot Analysis of Protein Markers
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Protein extracted using the preceding method was added to reduced Laemmli buffer (Bio-Rad), boiled for 5 min, and loaded into 4%–20% gradient polyacrylamide gels (Bio-Rad). Following electrophoresis, gel contents were transferred to a PVDF membrane (Millipore) and blocked with Odyssey blocking buffer (Li-Cor Biosciences) for 30 min. Membranes were then probed for proteins with the following primary antibodies: SMG1 (Dilution: 1:500; A300-394A, Bethyl Laboratories, Inc.) UPF1 (dilution: 1:1000; anti-RENT1, A301-902A, Bethyl Laboratories, Inc.), Grb2 (dilution: 1:1000; Upstate Cell Signaling), α-Tubulin (1:10,000; Novus Biologicals); and with the following secondary antibodies: goat anti-rabbit IRDye 680 (dilution: 1:10,000, Li-Cor Biosciences), goat anti-mouse IRDye 680 (dilution: 1:10,000, Li-Cor Biosciences), goat anti-mouse IRDye 800 (dilution: 1:10,000, Li-Cor Biosciences). All protein quantification was carried out using Odyssey's Image Studio version 3.0 (Li-Cor Biosciences).
6
Immunoblot Analysis of Connexin 43
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TGs (four per group) were removed after saline perfusion, homogenized, and protein extracted using the Trizol method (Invitrogen, Carlsbad, CA). Protein concentration was determined with bicinchroic acid (BCA) assay (Pierce, Rockford, IL). A protein aliquot of 30 μg was separated on 4–15% polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membrane. Membranes were incubated with Cx43 antibody (3512, Cell Signaling, Danvers, MA), followed by Anti-rabbit IRDye 680 (1:15,000, LI-COR, Lincoln, NE). Proteins were visualized with an Odyssey infrared scanner (LI-COR) and arbitrary optical density was determined. Normalizing controls were utilized by simultaneous staining with glyceraldehyde 3-phosphat dehydrogenase (GAPDH) antibody (1:1,000, WH0002597M1, Sigma, St. Louis, MO) followed by goat anti-mouse IRDye 800 (1:15,000, LI-COR). Protein levels were quantified via densitometry using NIH ImageJ Software.
7
Antibodies for Extracellular Vesicle Characterization
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The primary antibodies used in transmission electron microscopy and Western blotting and ELISA were as follows: mouse anti-CD63 (10628D; Invitrogen, Waltham, MA, USA); mouse-anti CD9 (AHS0902, Invitrogen, Waltham, MA, USA); and mouse anti-ApoB100/48 (3715-3-250, MabTech, Nacka Strand, Sweden). Antibodies anti-CD81 (10630D, Invitrogen, Waltham, MA, USA) and anti-Calnexin (C5C9) (#2679, Cell Signaling Technology, Danvers, MA, USA) were only used on ELISA.
The secondary antibody used in transmission electron microscopy (TEM) was gold-labelled (EM:GAM15, goat anti-mouse 15 nm immunogold-conjugate; BBI Solution, Crumlin, UK). For Western blotting, we used fluorescently labelled secondary antibodies: goat anti mouse IRDye® 800 or goat anti mouse IRDye® 680 or goat anti rabbit IRDye® 680 or goat anti rabbit IRDye® 800 (LI-COR Biosciences, Lincoln, NE, USA). The ELISA secondary antibodies were conjugated with horseradish peroxidase (HRP) as follows: Goat anti rabbit- HRP (111-036-047, Jackson Immunoresearch, West Grove, PA, USA); or Rabbit anti mouse-HRP (JZM035046_Fa, Ancell Immunology Research, Bayport, MN, USA).
The secondary antibody used in transmission electron microscopy (TEM) was gold-labelled (EM:GAM15, goat anti-mouse 15 nm immunogold-conjugate; BBI Solution, Crumlin, UK). For Western blotting, we used fluorescently labelled secondary antibodies: goat anti mouse IRDye® 800 or goat anti mouse IRDye® 680 or goat anti rabbit IRDye® 680 or goat anti rabbit IRDye® 800 (LI-COR Biosciences, Lincoln, NE, USA). The ELISA secondary antibodies were conjugated with horseradish peroxidase (HRP) as follows: Goat anti rabbit- HRP (111-036-047, Jackson Immunoresearch, West Grove, PA, USA); or Rabbit anti mouse-HRP (JZM035046_Fa, Ancell Immunology Research, Bayport, MN, USA).
8
Antibody Characterization and Validation
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The following antibodies were used: rabbit anti-HA (PRB-101P; Covance), rabbit anti-SPP (ab16080; Abcam), rabbit anti–hepatitis C core (R4210; gift from J. McLauchlan, Medical Research Council–University of Glasgow Centre for Virus Research, Glasgow, Scotland, UK), mouse anti–HO-1 (ab13248; Abcam), mouse anti-myc (9B11; Cell Signaling Technology), mouse anti–β-actin (A5316; Sigma-Aldrich), rabbit anti-RAMP4/4-2 (A18; sc-85114; Santa Cruz Biotechnology, Inc.), mouse anti-calnexin (AF8) and rabbit anti-GFP (A11122; Invitrogen), rabbit anti-US2 (177–5; ER luminal domain; a gift from E. Wiertz, University Medical Center Utrecht, Utrecht, Netherlands), mouse anti–MHC-I (W6/32; ATCC), goat anti–mouse Alexa 647 (Invitrogen), goat anti–mouse IRDye 800 and goat anti–rabbit IRDye 680 (LI-COR Biosciences), and goat anti–mouse HRP and goat anti–rabbit HRP (Jackson ImmunoResearch Laboratories, Inc.).
9
Pharmacological Modulation of AMPK Signaling
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A TSPO antibody was purchased from Thermo Fisher Scientific (PA5–75544, Shanghai, China). Antibodies against total AMPKα (#5232), phosphorylated AMPKα-172 (#50081), α-SMA (#19245), cyclin D1 (#2922), cyclin E (#4129), PCNA (#2586), H3 (#4499) and GAPDH (#5174) were obtained from Cell Signaling Technology (Beverly, MA, USA). Infrared-labeled donkey anti-rabbit IRDye 800 and goat anti-mouse IRDye 800 were purchased from Li-Cor Biosciences (Beijing, China). Secondary antibodies conjugated to Cy3 and FITC were purchased from Jackson Immuno-Research (West Grove, PA, USA). Cell culture dishes were obtained from NEST Biotechnology Co., Ltd (Shanghai, China). Different antagonists were used in this study, including protein kinase C (PKC) inhibitor peptide 19–31 (catalog no.: 05–23–4904, Calbiochem Co., Darmstadt, Germany), protein kinase A (PKA) inhibitor 14–22 amide (catalog no.: 476485, Calbiochem Co., La Jolla, CA, USA), the dihydropyridine calcium channel blocker nicardipine (catalog no.: N7510, Sigma Co., St. Louis, MO, USA), the mitogen-activated protein (MAP) kinase inhibitor PD98059 (catalog no.: P215, Sigma Co., St. Louis, MO, USA), and the PKC activator phorbol 12-myristate 13-acetate (PMA) (catalog no.: P8139, Sigma Co., St. Louis, MO, USA).
10
Western Blot Analysis of Protein Targets
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The following primary antibodies were used: anti-FLAG (Sigma-Aldrich, F1804), anti-HA (Sigma-Aldrich, H6908), anti-GFP (Sigma-Aldrich, G1546), anti-GAPDH (Sigma-Aldrich, G8796), anti-β-tubulin (Sigma-Aldrich, T6199), anti-ORF57 antibodies were described previously [18 (link)]. The secondary antibodies were as follows: goat anti-mouse IRDye800 (LI-COR, 926–32210), goat anti-rabbit IRDye800 (LI-COR, 926–32211), goat-anti-mouse-horseradish peroxidase (HRP) (Sigma-Aldrich, A2554). All protein samples were denatured by SDS-loading buffer and separated by SDS-polyacrylamide gel and transferred to nitrocellulose (NC) membrane, the individual proteins were stained by appropriate antibodies and detected by ODYSSEY (LI-COR) or SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
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