Total proteins were extracted from isolated EVs by lysing samples with ice-cold RIPA buffer supplemented with SIGMAFAST™ Protease Inhibitors and Phosphatase Inhibitor Cocktail 3 and 2 (all from Sigma). Proteins were boiled with Laemmli SDS sample buffer 6X (VWR International), separated on 4–20% MiniPROTEAN®TGX™ Precast Gel, and transferred onto a PVDF membrane with a semi-dry transfer system (all from Bio-Rad Europe, Basel, Switzerland). Membranes were incubated with ALIX (Abcam ab186429; 1:1000), TSG101 (Abcam ab125011, 1:1000), and Syntenin-1 (Abcam ab19903; 1:1000) and Apolipoprotein A1 (APOA1) (Invitrogen 701239, 1:500) primary antibodies. Secondary antibodies IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit (LI-COR Biosciences) were used for detection. The infrared signal was detected using Odyssey CLx Detection System (LI-COR Biosciences).
Odyssey clx detection system
Manufactured by LI COR
Sourced in United States
The Odyssey CLx Detection System is a fluorescence-based imaging system designed for the quantitative analysis of proteins and nucleic acids. It utilizes infrared fluorescence technology to enable high-sensitivity and high-resolution detection of multiple targets within a single experiment. The system provides accurate and reproducible data for a wide range of life science applications.
Automatically generated - may contain errors
Lab products found in correlation
Mini protean tgx precast gel, by Bio-Rad (3 mentions)
Laemmli sds sample buffer 6x, by Avantor (3 mentions)
Irdye 680rd, by LI COR (3 mentions)
Sigmafast protease inhibitors and phosphatase inhibitor cocktail 3 and 2, by Merck Group (2 mentions)
800cw goat anti mouse or goat anti rabbit, by LI COR (2 mentions)
Semi dry transfer system, by Bio-Rad (2 mentions)
Anti grp94, by Abcam (1 mentions)
Anti periostin, by Santa Cruz Biotechnology (1 mentions)
Anti tsg101, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Ab290, by Abcam (1 mentions)
M4439, by Merck Group (1 mentions)
A0480, by Merck Group (1 mentions)
Rabbit monoclonal anti tsg101, by Abcam (1 mentions)
Sds page 4 12 gel, by Bio-Rad (1 mentions)
Rabbit monoclonal anti alix, by Abcam (1 mentions)
Irdye 800cw goat anti rabbit secondary antibody, by LI COR (1 mentions)
Odyssey clx analyzer, by LI COR (1 mentions)
G actin f actin in vivo assay biochem kit, by Cytoskeleton (1 mentions)
Ab26300, by Abcam (1 mentions)
8 protocols using odyssey clx detection system
1
Extracellular Vesicle Protein Profiling
2
Exosomal Protein Analysis by Western Blot
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Total proteins were extracted by lyzing exosomes or cells in the presence of protease inhibitors. Proteins were separated on gel and transferred onto a PVDF membrane. Anti-Periostin (Santa Cruz, United States), anti-TSG101, anti-GRP94, and anti-GAPDH (Abcam, United Kingdom) were used as primary antibodies; IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit (LI-COR Biosciences, United States) as secondary antibodies. The infrared signal was detected using the Odyssey CLx Detection System (LI-COR Biosciences).
3
Immunoblotting Analysis of Plant Proteins
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Total proteins were extracted from rosettes homogenized in 50 mM Tris–HCl, pH 8, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM DTT, 1× Complete Protease Inhibitor EDTA-free Mixture (Roche). Equal amounts of soluble proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and subject to immunoblotting. For stable lines expressing YFP-tagged proteins, anti-GFP/YFP (rabbit, ab290; Abcam, Cambridge, UK) and anti-plant actin (mouse, A0480; Sigma-Aldrich) were used. For immunoblotting of BAM9, we used antibodies raised in rabbits (Eurogentec, Seraing, Belgium) against the recombinant purified protein. For immunoblotting of BAM3, we used antibodies described previously (Fulton et al., 2008) (link). Proteins were detected using near-infrared fluorescence from IRDye secondary antibodies (anti-rabbit and/or anti-mouse) and an Odyssey CLx detection system (Li-Cor, Lincoln, NE, USA). For the stable BAM9-TAP lines, an anti-myc (mouse, M4439; Sigma-Aldrich) antibody was used to detect the proteins followed by colorimetric detection.
4
Extracellular Vesicle Protein Profiling
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WB was performed on protein lysate after EV bead-based immuno-capture. Serum aliquots were incubated overnight with MACSPlex capture beads and saline solution. Unbounded fraction was discarded, and samples were lysed in RIPA buffer; total proteins were separated on SDS Page 4–12% gel (BioRad) and signals were detected by Odyssey CLx Detection System (LI-COR Biosciences). Blots for 3 representative samples were incubated with the following primary antibodies: rabbit polyclonal anti-ApoB48, mouse monoclonal anti-GRP94, rabbit monoclonal anti-Alix, rabbit monoclonal anti-CD142, rabbit monoclonal anti-TSG101, rabbit polyclonal anti-Syntenin-1, rabbit monoclonal anti-CD81 (all from Abcam), and rabbit monoclonal anti-Mitofillin (Invitrogen).
5
Cytoskeleton Rearrangement in mNVCM after hAFSC Secretome Stimulation
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To investigate mNVCM cytoskeleton re-arrangement following stimulation with the hAFSC secretome over vehicle solution as control, the amount of polymerized F-Actin over monomeric G-Actin was analyzed as indication of cardiomyocyte cell cycle progression (Torrini et al., 2019 (link)). mNVCM were seeded on a 24-well plate as 500.000 cells/well, which were then stimulated by 5 μg/ml hAFSC-EVs (corresponding to 2 μg per well in 400 μl of medium final volume). The F-Actin/G-Actin In Vivo Assay Biochem Kit (Cytoskeleton) was used as per manufacturer’s instructions. mNVCM proteins were boiled with Laemmli SDS sample buffer 6x (VWR International), separated on 4–20% Mini-PROTEAN®TGX™ Precast Gel, and transferred onto a polyvinylidene difluoride (PVDF) membrane with a semi-dry transfer system (all from Bio-Rad). Membrane were incubated with F-Actin/G-Actin antibody provided in the assay and then with IRDye® 800CW goat anti-rabbit secondary antibody (LI-COR Biosciences). Infrared signals were detected using the Odyssey CLx Detection System (LI-COR Biosciences). Quantification was performed using an Odyssey CLx analyzer (LI-COR Biosciences).
6
Western Blot Analysis of MSC-Derived Extracellular Vesicles
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For Western blot analysis, the pre-conditioned MSCs and their corresponding separated mEVs and sEVs were resuspended in RIPA buffer (1% NONIDET p-40, 0.1% SDS, 0.1% Sodium deoxycholate, protease inhibitor cocktail 1x, in PBS pH 7.5) and protein content was quantified by BCA assay. Afterwards, 10 μg of proteins for each sample was loaded on 4-20% Mini-PROTEAN®TGX™ Precast Gel for electrophoresis, and proteins were blotted onto a PVDF membrane with a semi-dry transfer system (all from Bio-Rad Europe, Basel, Switzerland). Blot membrane was incubated overnight at 4 • C with the following specific primary antibodies: anti-syntenin (1:1000 dilution, ab133267 Abcam, USA), anti-mitofilin (1:1000 dilution, PA5 89627, Invitrogen, USA) and anti-Grp94 (1:1000 dilution, ab238126, Abcam, USA), and anti-lamin A (1:1000 dilution, ab26300, Abcam, USA) prepared in odyssey blocking buffer (LI-COR, USA) at the dilution as recommended by the manufacturer. Following, membranes were incubated with appropriate secondary antibodies such as IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit secondary Ab (LI-COR Biosciences, Lincoln, Nebraska, USA). Infrared signal was detected using Odyssey CLx Detection System (LI-COR Biosciences).
7
Proteomic Profiling of Extracellular Vesicles
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Total proteins were extracted by lysing samples with ice-cold RIPA buffer supplemented with SIGMAFAST™ Protease Inhibitors and Phosphatase Inhibitor Cocktail 3 and 2 (all from Sigma, St. Louis, MI, USA). Protein concentration was determined using BCA (Thermo Fisher Scientific). Proteins were boiled with Laemmli SDS sample buffer 6x (VWR International, Dietikon, Switzerland), separated on 4–20% Mini-PROTEAN®TGX™ Precast Gel, and transferred onto a PVDF membrane with a semi-dry transfer system (all from Bio-Rad Europe, Basel, Switzerland). Membranes were incubated with appropriate antibodies (TF-CD142 ab252918; CD81 ab109201; Syntenin-1 ab19903; TSG101 ab125011; Apo-B48 ab20737; Apo-A1 ab33470 all from aBCAm) and then with IRDye® 680RD or 800CW goat anti-mouse or goat anti-rabbit secondary Ab (LI-COR Biosciences, Lincoln, Nebraska, USA). Infrared signal was detected using Odyssey CLx Detection System (LI-COR Biosciences). Full images of membranes are provided in Fig. S1b.
8
Western Blot Analysis of Cellular Proteins
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Total proteins were extracted by lysing cells and then heated at 95 °C for 5 min (except for SERCA2 and PLN detection) with Laemmli SDS sample buffer 6× containing: 0.375 M Tris-HCl pH 6.8, 12% SDS, 60% glycerol, 0.6 M DTT, 20% (v/v) beta-mercaptoethanol, 70.2% (w/v) bromophenol blue (VWR International LCC). Proteins were separated on 4-20% Mini-PROTEAN® TGX™ Precast Gel (Bio-Rad) (or 4-12% Bis-Tris Criterion BIO-RAD gels for SERCA2 and PLN detection) and transferred onto a PVDF membrane with a semi-dry transfer system (Bio-Rad). The membranes were blocked for 1 h with Intercept (TBS) Blocking Buffer (Licor) or milk and incubated with the primary Abs at 4 °C overnight (anti-p16, 1:1'000, Proteintech 10883; anti-p21, 1:1'000 eBiosciences 14-6715-81; anti-KCNH2, 1:200, Alomone APC-109; anti-P AMPK, 1:1'000, Cell Signaling 2537; anti-CAMKII, 1:2'000, Abcam 92332; anti-P CAMKII, 1:2'000, Abcam 171095; anti-SERCA2, 1:1000, N-19 Santa Cruz Biotechnology; anti-PLN 1:1000, 2D12, Abcam; anti-actin 1:5000, Merck). Membranes were then rinsed and incubated with appropriated fluorophore-conjugated secondary antibodies (Li-COR) at RT for 2 h. Subsequently, the membranes were rinsed then acquired and analyzed using the Odyssey CLx Detection System (LI-COR Biosciences).
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