1.5 ml of eluted EVs were concentrated 8-fold by centrifugation at 2000 g for 20 min at 4°C, using a 100 kDa cutoff centricon (MerckMillipore, Darmstadt, Germany). THP1 cells, used as positive control, were grown in RPMI containing 10% FCS and P/S (14 (link)). Cell lysates were prepared in RIPA buffer (50 mmol/l Tris-HCl pH 8, 150 mmol/l NaCl, 1% Nonidet P-40, 0.1% SDS, 1% Triton X-100 plus proteinase inhibitors). Concentrated EVs or 30 µg of THP1 cell lysates were resolved in 10% SDS-polyacrylamide gels under non-reducing conditions and electrophoretically transferred to nitrocellulose membranes (Bio-Rad Laboratories). These were then blocked with Starting Block TBS buffer (Thermo Fisher) for 1 h at rt and incubated overnight at 4°C with anti-CD9 (Immunostep) or CD63 mAb (RRID : AB_396297) diluted in blocking buffer. The membranes were subsequently incubated for 60 min at rt with IRDye 800Cw-conjugated goat anti-mouse IgG (LI-COR Biosciences Lincoln, NE) (RRID : AB_621842) diluted in blocking buffer. Three 15-min washes between steps were performed with TBS-0.01% Tween 20 (Merck Millipore). Bound antibody was detected with an Odyssey Infrared Imager (LI-COR), and densitometric analysis was performed using the Odyssey V.3 software (LI-COR).
Irdye 800cw conjugated goat anti mouse igg
Manufactured by LI COR
Sourced in United States
IRDye 800CW-conjugated goat anti-mouse IgG is a fluorescent-labeled secondary antibody. It is designed to detect and quantify mouse immunoglobulin G (IgG) in various applications, such as Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Odyssey system, by LI COR (4 mentions)
Odyssey infrared imaging system, by LI COR (4 mentions)
Irdye 680 conjugated goat anti rabbit igg, by LI COR (3 mentions)
Odyssey infrared imager, by LI COR (2 mentions)
Complete ultra tablet, by Roche (2 mentions)
Pdvf membrane, by Merck Group (2 mentions)
Odyssey blocking solution, by LI COR (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Irdye 800cw conjugated goat anti rabbit igg, by LI COR (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti cd9, by Immunostep (1 mentions)
Odyssey v3, by LI COR (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Mouse anti α tubulin 12g10, by Developmental Studies Hybridoma Bank (1 mentions)
Mouse anti ha 16b12, by BioLegend (1 mentions)
Rabbit anti phospho creb ser133 antibody, by Cell Signaling Technology (1 mentions)
Mouse α tubulin antibody, by Abcam (1 mentions)
15 protocols using irdye 800cw conjugated goat anti mouse igg
1
Extracellular Vesicle Characterization by Western Blot
2
Western Blotting for Parasite Protein Analysis
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Freshly egressed parasites were filtered and washed in PBS before lysis in 2× Laemmli buffer (4% SDS, 20% glycerol, 5% 2-mercaptoethanol, 0.02% bromophenol blue, 120 mM Tris-HCl, pH 6.8). Samples were heated to 100°C for 5 min prior to resolution by SDS-PAGE. After transferring the separated proteins onto nitrocellulose at a constant 25 V overnight, membranes were blocked for 1 h at room temperature in TBS-T, 5% (wt/vol) nonfat dry milk. Immunoblots were probed, as indicated, with rabbit antialdolase (anti-ALD) (WU1614 [39 (link)]) diluted 1:2,000, mouse anti-α-tubulin (12G10; Developmental Studies Hybridoma Bank) diluted 1:10,000, mouse anti-FLAG (M2; Sigma-Aldrich) diluted 1:5,000, mouse anti-CAT (MAB3678; EMD Millipore monoclonal) diluted 1:1,000, rabbit anti-actin (TgACT1) (40 (link)) diluted 1:10,000, mouse anti-Ty (BB2 [41 (link)]) diluted 1:5,000, mouse anti-2A peptide (3H4; Novus Biologicals) diluted 1:1,000, or mouse anti-HA (16B12; BioLegend) diluted 1:2,000. The signal was detected using 1:20,000 dilutions of IRDye 800CW-conjugated goat anti-mouse IgG and IRDye 680CW-conjugated donkey anti-rabbit IgG (Li-Cor Biosciences) on an Odyssey infrared imager (Li-Cor Biosciences).
3
Western Blot Analysis of cTnT, CREB, and Tubulin
4
Immunoblot Analysis of p-Smad1/5/8
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Cells were lysed in RIPA cell lysis buffer (Santa Cruz, Santa Cruz, CA) supplemented with protease inhibitor cocktail (Santa Cruz) and phosphatase inhibitor cocktail 2 (Santa Cruz). Protein concentrations were determined by using BCA protein kit (Thermo Fisher), and cell lysate was isolated in SDS-PAGE and transferred onto PVDF membrane. Alpha-tubulin and p-Smad1/5/8 were detected by Odyssey system (Li-Cor bioscience) following incubation with the appropriate primary and secondary antibodies. Primary antibodies used were rabbit anti-p-Smad1/5/8 (Cell Signaling Tech, 1∶1000 dilution) and mouse anti-alpha-tubulin (Cell Signaling Tech, 1∶1000 dilution). The secondary antibodies used include IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience, 1∶5000 dilution) and IRDye 800CW-conjugated goat anti-mouse IgG (Li-Cor Bioscience, 1∶5000 dilution).
5
Western Blot Analysis of Smad Signaling
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Cells were lysed with RIPA buffer (Sigma) containing protein inhibitors (complete ULTRA Tablets, Roche, Basel, Switzerland) and phosphatase inhibitors (PhosSTOP, Roche, Basel, Switzerland). Samples were denatured by incubating at 95 °C for 5 min in sample buffer and separated by using SDS-PAGE (precast 8–16% gradient gels, Biorad, Hercules, CA, USA). Then, the samples were transferred to a PDVF membrane (Millipore). The membrane was blocked with Odyssey Blocking solution (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature, followed by primary antibody incubation at 4 °C overnight. The primary antibodies used in the present study included rabbit anti-p-Smad1/5/9 (Cell Signaling Tech, Danvers, MA, USA), rabbit anti-Smad 1(Cell Signaling Tech), mouse anti-beta actin (Cell Signaling Tech), rabbit anti-p-Smad 2 (Cell Signaling Tech) and rabbit anti-Smad 2 (Cell Signaling Tech). The proteins were detected by Odyssey system (Li-Cor bioscience) followed by the secondary antibodies including IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience) and IRDye 800CWconjugated goat anti-mouse IgG (Li-Cor Bioscience, Lincoln, NE, USA).
6
Western Blot Analysis of FXIIIa in BMCMCs
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BMCMCs were isolated and cultured as described previously (16 (link)). Cells were pelleted, and proteins were extracted in M-PER lysis buffer supplemented with a protease inhibitor cocktail (Thermo Fisher) on ice for 10 minutes as described (73 (link)). Protein content of cell lysates was determined by BCA assay, and 12 μg protein was resolved on NuPAGE Bolt Bis-Tris 4 to 12% gels (Fisher Scientific). Proteins were then transferred to a nitrocellulose membrane, and the blots were probed with the following antibodies: mouse anti-FXIIIa (1:1,000, catalog AC-1A1, Abcam) or control rabbit anti–β-actin (1:5,000, clone 13E5, Cell Signaling Technology), followed by 3 × 5 minutes washes with TBS-T buffer. Secondary antibodies used were IRDye 800CW-conjugated goat anti-mouse IgG (1:10,000, LI-COR Biosciences, catalog P/N: 926-32210) or IRDye 680LT-conjugated goat anti-rabbit IgG (1:10,000, LI-COR Biosciences, catalog P/N: 926-68021) for 30 minutes. Western blots were imaged on a LI-COR Odyssey CLx (LI-COR Biosciences), and densitometry analysis was performed using Image J.
7
Membrane Protein Expression Analysis
8
SDS-PAGE Analysis of Tri-S Protein
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Recombinant tri-S protein was heat-denatured at 100°C for 3 min in loading buffer (Invitrogen) containing 1× sample reducing agent (Invitrogen). Denatured tri-S protein (50 µg total) was separated by SDS-PAGE with a NuPAGE 4–12% Bis-Tris Gel (1-well; Invitrogen), electro-transferred onto nitrocellulose membranes, and saturated in PBS-0.05% Tween 20 (PBST)-5% dry milk overnight at 4°C. Membranes were inserted into a Miniblot apparatus (Immunetics) and then incubated with human mAbs (at a concentration of 1 µg/ml) and mouse anti-6xHis antibody (1 µg/ml; BD Biosciences) in PBS-T 5% dry milk in each channel for 2 h. For dot blotting experiments, denatured tri-S (ranging from 0.125 to 2 µg) was immobilized on dry nitrocellulose membranes for 2 h at room temperature and saturated in PBS-0.05% Tween 20 (PBST)-5% dry milk overnight at 4°C. The membranes were then incubated with human mAbs (at a concentration of 1 µg/ml) and mouse anti-6xHis antibody (1 µg/ml; BD Biosciences) in PBS-T 5% dry milk for 2 h. After washing with PBST, membranes were incubated for 1 h with 1/25,000-diluted Alexa Fluor 680-conjugated donkey anti-human IgG (Jackson ImmunoResearch) and 1/25,000-diluted IR Dye 800CW-conjugated goat anti-mouse IgG (LI-COR Biosciences) in PBST-5% dry milk. Finally, membranes were washed and examined with the Odyssey Infrared Imaging system (LI-COR Biosciences).
9
Immunoblotting of Nef Expression in HEK293T Cells
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HEK293T cells were cultured in 6-well plates and transfected with plasmids expressing Nef as described above. Two days post-transfection, HEK293T cells were lysed in RIPA-buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) containing Complete EDTA-free protease inhibitor (Roche, Basel, Switserland). After adding NuPAGE LDS 4× sample buffer (Invitrogen, Carlsbad, CA, USA) and 0.1 M dithiothreitol (DTT; Invitrogen, Carlsbad, CA, USA), samples were heated at 95 °C for 10 min. Proteins were separated by SDS-PAGE (NuPAGE 4–12% Bis-Tris precast gel and MES SDS running buffer (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane (Protran, Schleicher and Schuell, Dassel, Germany) using NuPAGE transfer buffer (Invitrogen, Carlsbad, CA, USA). After blocking for 2 h with PBS containing 5% Protifar (Nutricia, Schiphol, The Netherlands) and 0.5% BSA, the blot was incubated with anti-c-Myc antibody (1∶5000; Calbiochem, San Diego, CA, USA) and anti-β-actin antibody (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA, USA). IRDye 800CW conjugated goat anti-mouse IgG (1∶15000, LI-COR, Lincoln, NE, USA) and IRDye 680LT conjugated donkey anti-goat IgG (1∶15000, LI-COR) were used as secondary antibodies to visualize expression using the proteins.
10
Western Blot Protein Detection
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Cells were lysed with RIPA buffer (Sigma-Aldrich, St Louis, MO, USA) containing protein inhibitors (complete ULTRA Tablets, Roche) and phosphatase inhibitors (PhosSTOP, Sigma-Aldrich, St Louis, MO, USA). Samples were denatured by incubating at 95 °C for 5 min in sample buffer and separated by using SDS-PAGE gels). Then the samples were transferred to a PDVF membrane (Millipore, Burlington, MA, USA). The membrane was blocked with Odyssey Blocking solution (Li-Cor Biosciences, Lincoln, NE, USA) for 1 h at room temperature, followed by primary antibody incubation at 4 °C overnight. The primary antibodies used in the present study included rabbit anti-PDE4D (Cell Signaling Tech, Danvers, MA, USA), mouse anti-beta actin (Cell Signaling Tech, Danvers, MA, USA). The proteins were detected by Odyssey system (Li-Cor bioscience, Lincoln, NE, USA) followed by the secondary antibodies including IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience) and IRDye 800CWconjugated goat anti-mouse IgG (Li-Cor Bioscience, Lincoln, NE, USA).
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