Cells were lysed in an LDS sample buffer (NuPAGE, Novex, Life Technologies, Carlsbad, CA, USA), sonicated, and boiled at 96 °C for 3 min. Proteins were separated on 4–20% gradient SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Burlington, MA, USA). Membranes were blocked in TBS-T (Tris Buffered Saline with 0.1% Tween 20) containing 5% Bovine Serum Albumin (Euromedex, Souffelweyersheim, France) or 5% skim milk and incubated with primary antibodies overnight at 4 °C. Membranes were washed and then incubated at room temperature with Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Ely, UK). After washing, detection was performed by chemiluminescence (ClarityTM Western ECL substrate, Bio-Rad). Image acquisition and quantification of immunoblots were performed with a Fusion Solo X chemiluminescence imaging system using the Evolution Capture software (Vilber Lourmat, Marne La Vallée, France).
Fusion solo x
Manufactured by Vilber
Sourced in France, United States
The Fusion Solo X is a compact and versatile imaging system designed for DNA and protein gel documentation. It features a high-resolution camera and adjustable LED light sources for capturing clear and accurate images of electrophoresis gels.
Automatically generated - may contain errors
Lab products found in correlation
Ecl substrate, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Bioss Antibodies (2 mentions)
Bovine serum albumin (bsa), by Euromedex (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Evolution capture software, by Vilber (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Claritytm western ecl substrate, by Bio-Rad (1 mentions)
Evolution capt software, by Vilber (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti flag, by Merck Group (1 mentions)
Biotinylated maackia amurensis lectin 1 mal 1, by Vector Laboratories (1 mentions)
Neu a, by New England Biolabs (1 mentions)
Neu s, by New England Biolabs (1 mentions)
Biotinylated sambucus nigra agglutinin sna, by Vector Laboratories (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence detection system, by GE Healthcare (1 mentions)
Anti bmp2, by Abcam (1 mentions)
Anti ocn, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
10 protocols using fusion solo x
1
Western Blot Protein Detection
2
Western Blot Analysis of VLPs
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Concentrated VLPs and infected cell lysates were mixed with SDS solubilizer to final concentrations of 0.0625 M Tris-HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, 2% (w/v) SDS, and 1% 2-mercaptoethanol. Samples were heated at 95 °C for 5 min, separated in 10% (w/v) polyacrylamide-SDS gels, transferred to PVDF membranes, probed with monoclonal mouse anti-C9 (Santa Cruz) or monoclonal mouse anti-Myc antibody (Santa Cruz, Dallas, TX, USA). Membranes were then probed with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss, Woburn, MA, USA), developed with ECL substrate (Thermo Fisher Scientific, Middlesex, MA, USA), and signals were detected using Fusion Solo X (Vilber, France). Band density on western blot membranes was analyzed using Evolution Capt software (Vilber, France) (shown in Figure S1 ).
3
Glycan Profiling of Nanoparticles
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Nanoparticles were incubated with neuraminidase A (Neu A, New England biolabs, Inc., Beverly, MA, USA), neuraminidase S (Neu S, New England biolabs, Inc.), or PNGase F (New England biolabs, Inc.) at 37 °C for 18 h. Proteins were transferred to a polyvinylidene fluoride membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking with 5% (w/v) bovine serum albumin in Tris-buffered saline with 0.05% TWEEN 20 (TBST) for 1 h at 25 °C, membranes were probed with monoclonal mouse anti-flag (1:1000, Sigma-Aldrich, St. Louis, MO, USA) antibodies, biotinylated Sambucus nigra agglutinin (SNA, Vector Laboratories, Burlingame, CA, USA), or biotinylated Maackia amurensis lectin-I (MAL-I, Vector Laboratories, Burlingame, CA, USA) as a primary staining, and then with horseradish peroxidase-conjugated goat anti-mouse IgG (Bioss Antibodies, Woburn, MA, USA) and streptavidin at 1:5000 dilution in TBST. Membranes were developed using ECL substrate (Thermo Fisher Scientific, Waltham, MA, USA) and signals were detected with Fusion Solo X (Vilber, Paris, France).
4
Western Blot Analysis of Osteogenic Markers
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Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 4% skim milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature. Proteins were detected with primary antibodies anti-DMP1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-RUNX2 (MBL, Woburn, MA, USA), anti-BMP2 (Abcam, Cambridge, UK), anti-OCN (Abcam), and anti-β-actin (Santa Cruz Biotechnology), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Enzo Life Sciences, Minneapolis, MN, USA). It was detected using an enhanced chemiluminescence detection system (GE Healthcare, Buckinghamshire, UK), according to the manufacturer's instructions. Signals were detected using a Fusion Solo X (Vilber, Paris, France). The protein expression levels were normalized to that of β-actin. All Western blot analyses were repeated three times under the same conditions.
5
MERS-CoV Structural Protein Detection
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Transfected cells were lysed in Triton X-100 lysis buffer (1% Triton X-100, 50 mM Tris–Cl [pH 8.0], 150 mM NaCl, 1 mM EDTA) on ice and cleared by centrifugation at 1,000 × g for 10 min at 4 °C. The concentrated VLPs and transfected cell lysates were mixed with SDS solubilizer. Samples were heated at 95 °C for 10 min, separated in 8% or 15% (wt/vol) polyacrylamide-SDS gels, transferred to PVDF membranes, and probed with polyclonal mouse anti-MERS-CoV S (Sino Biological, 1:1000), polyclonal rabbit anti-MERS-CoV M (GeneTex, 1:1000), polyclonal rabbit anti-MERS-CoV E (GeneTex, 1:1000), or monoclonal mouse anti-myc (Santa Cruz, 1:1000) antibodies. The membranes were then probed with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Promega, 1:1000) or anti-rabbit IgG (Bioss, 1:1000) and incubated with ECL substrate (Thermo Fisher Scientific), and the signals were detected using a Fusion Solo X (Vilber). Band density on western blot membranes was analyzed using Evolution Capt software.
6
Cytosolic and Nuclear Protein Extraction for Western Blotting
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Western blotting was performed as described previously (25 (link)). The cytosolic and nuclear fraction preparation was performed using ExKine™ Nuclear and Cytoplasmic Protein Extraction Kit (Abbkine, China) according to the manufacturer’s instructions. The primary antibodies used were as follows: anti-NLRP3 (1:1,000; Cell Signaling, Danvers, MA, USA), anti-XIAP (1:1,000; Enzo Life Sciences, Farmingdale, NY, USA), anti-ASC (1:1,000; Abbkine), anti-caspase-1 (1:1,000; Novus Biologicals), anti-TLR4 (1:1,000; Bioworld Technology), anti-myeloid differentiation factor88 (MyD88, 1:1,000; Cell Signaling), anti-NF-κB (1:1,000; Cell Signaling), anti-Lamin B1 (1:1,000; Cell Signaling), anti-β-tubulin (1:1,000; Abbkine), and anti-β-actin (1:5,000; Enzo Life Sciences). The membranes were then washed thrice with Tris-buffered saline with Tween-20 (TBST) and incubated with secondary antibodies conjugated with horseradish peroxidase. Immunopositive bands were enhanced with chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA) and visualized using the Fusion Solo X (Vilber Lourmat, Collegien, France).
7
Western Blot Analysis of Osteogenic Proteins
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The cellular proteins were extracted using 1 × RIPA buffer from cell pellets and the concentration of cellular proteins was measured using a BCA protein assay kit. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. After blocking with 5% skim milk in TBS-T (Tris-buffered saline containing 0.1% Tween-20), the membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies against ALP, OPN, Runx2, Osterix, BMP-2, Smad-1, p38, ERK, JNK, β-actin (1:5000 dilution), phospho-Smad1, phospho-p38, phospho-ERK, phospho-JNK (1:3000 dilution), PPAR gamma, c-Fos, and NFATc1 were used from Santa Cruz Biotechnology (San Diego, CA, USA). Next, the nitrocellulose membranes were primed with secondary antibodies (Anti-Rabbit or Anti-Mouse with horseradish peroxidase). Immunoreactive protein bands were detected using a chemiluminescent reagent (Thermo Fisher Scientific). Relative protein images were captured using Fusion SOLO X (Vilber, Marne-la-Vallée, France).
8
Osteogenic Differentiation Protein Expression
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ADSC before osteogenic induction (day 0) and ADSC after osteogenic media treatment (day 3 and day 6) were lysed to obtain cell lysates. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL, USA). Protein samples were denatured, electrophoresed by SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5 % skim milk or 5 % BSA, the membranes were incubated with primary antibodies for c-Met, P-Met, FGF2, Runx-2, osterix, ALP, and GAPDH, followed by anti-immunoglobulin G horseradish peroxidase-conjugated secondary antibody. Blots were developed and visualized using WESTAR ECL Substrates (Cyanagen, Bologna, Italy) and analyzed by chemiluminescence using Fusion Solo X (Vilber Lourmat, Marne-La-Vallée, France). The expression levels were quantified using ImageJ software.
9
Immunoblotting Analysis of CGT Protein
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SDS-PAGE and Western blotting were performed as described previously [38 (link)]. The following antibodies were used in this study: rabbit anti-CGT (dilution 1:4000; kind gift from Gerrit van Meer) [39 (link)], rabbit anti-α-tubulin (dilution 1:20,000; cat# 600-401-880, Rockland Immunochemicals, Limerick, PA, USA), and peroxidase-conjugated goat anti-rabbit IgG (dilution 1:20,000; cat# 111-035-003, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Bound secondary antibodies were detected by enhanced chemiluminescence using Pierce ECL Western blotting substrate (Thermo Fisher, Waltham, MA, USA) and a CCD camera system (Fusion Solo X; Vilber Lourmat, Eberhardzell, Germany).
10
Protein Quantification and Western Blotting
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Sampled tissues were mechanically disrupted in RIPA buffer (pH 8.0) supplemented with a protease inhibitor cocktail (Complete Mini, Roche Diagnostics, Grenzach-Wyhlen, Germany). Protein concentrations were determined using the PierceTM BCA Protein Assay kit (Thermo Fisher Scienti c, Waltham, USA) according to the manufacturer's protocol. Per sample, a total of 20 µg protein was separated in a 14% SDS polyacrylamide gel by gel electrophoresis and transferred to a PVDF (polyvinylidene di uoride) membrane. The blots were blocked in 5% milk in Tris-buffered saline (TBS, pH 7.4) and then incubated overnight (at 4°C) in primary antibodies rabbit anti-Lcn2 in 5% milk and rabbit anti-GAPDH in 5% milk (used antibodies are listed in table 2). An appropriate secondary antibody (goat anti-rabbit IgG (H + L)-HRP) was applied for 2 h (RT). Signals were analyzed via chemiluminescence detection (Westar Supernova, XLS 3,0100, Cyanagen, Bologna, Italy), visualized (Fusion Solo X, Vilber, Eberhardzell, Germany) and subjected to densitometry analysis using Image J. Results were normalized to GAPDH as reference protein.
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