To detect intact RopB protein extracted from R. leguminosarum cells, rabbit anti-RopB (PrimeBioMed LLC, Moscow, Russia) antibody and secondary goat anti-rabbit IgG (H+L) antibody (Thermo Scientific, Waltham, MA, USA) were used in the dilutions 1:1,000 and 1:20,000, respectively. Visualization of protein signals by the enhanced chemiluminescence (ECL) PrimeWestern Blotting Detection reagent (GE Healthcare, Chicago, IL, USA), and Bio-Rad ChemiDoc™ hardware and software (Bio-Rad, Hercules, CA, USA) was used for imaging. All samples at the appropriate stage of sample preparation (before adding detergents) underwent concentration equalization near the value of 1 mg/mL of total protein. Measurements were taken using the Qubit 3.0 Fluorometer (Invitrogen, Carlsbad, CA, USA).
Prime western blotting detection reagent
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The Prime Western Blotting Detection Reagent is a lab equipment product designed for the detection and visualization of target proteins in Western blotting experiments. It provides a reliable and efficient method for detecting and quantifying proteins of interest.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Skim milk powder, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Rabbit anti β actin, by Cell Signaling Technology (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Imagequant tl, by GE Healthcare (2 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Mouse anti β actin monoclonal antibody, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Rat anti α tubulin, by Abcam (1 mentions)
Rabbit anti gfp, by Cell Signaling Technology (1 mentions)
Rabbit anti tdp43, by Cell Signaling Technology (1 mentions)
Amersham imager 600, by Cytiva (1 mentions)
Seeblue 2, by Thermo Fisher Scientific (1 mentions)
Penta his hrp conjugate kit, by Qiagen (1 mentions)
Goat anti icam1, by R&D Systems (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
19 protocols using prime western blotting detection reagent
1
Detecting Bacterial RopB Protein
2
Comparing CADM1 Expression in GIST Subtypes
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Equals of protein samples extracted from fresh frozen tissues of eight small intestinal (jejuno-ileal) GISTs with exon 11 c-kit mutation and eight gastric GISTs with exon 11 c-kit mutation were separated by 12% SDS-PAGE and subsequently transferred to PVDF membrane (Invitrogen, Carlsbad, CA). Membrane was incubated with anti-CADM1 chicken monoclonal antibody (MBL International, Woburn, MA) diluted at 1:1,000 for 3 h at room temperature using iBindTM Western System (Invitrogen). Immunoreactive protein bands were visualized using Prime Western Blotting Detection Reagent (GE Healthcare Life Science, Buckinghamshire, United Kingdom) on ChemiDoc Imaging System (Bio-Rad, Laboratories, California, United States). Reprobing by anti-KIT rabbit polyclonal antibody (DAKO, Carpinteria, CA) and anti-β-actin mouse monoclonal antibody (Abcam, Cambridge, United Kingdom) was done.
3
Western Blot Analysis of sEV Proteins
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Protein concentrations in sEV preparations were determined by a standardized bicinchoninic acid (BCA) assay according the manufacturer’s protocol (Pierce, Rockford, IL, U.S.A.). For Western blot, 30 µg protein samples were solubilised with Laemmli sample buffer under reducing (containing dithiothreitol [DTT]; AppliChem, Darmstadt, Germany) or non-reducing (not containing DTT) conditions and separated on SDS–PAGE gels before transfer to polyvinylidene fluoride membranes (PVDF; Millipore, Darmstadt, Germany). Membranes were blocked in TBS-T supplemented with 5% (w/v) skim milk powder (Sigma-Aldrich). Membranes were stained with rabbit anti-syntenin (clone EPR8102; Abcam), rabbit anti-calnexin (ab10286; Abcam) or mouse anti-CD9 (clone VJ1/20.3.1; kindly provided by Francisco Sánchez, Madrid, Spain) antibodies. Anti-syntenin and anti-CD9 antibodies were used as sEV markers, anti-calnexin antibody as cellular contamination marker. Membranes were washed and counterstained with appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) that were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science).
4
Neutrophil Histone Citrullination Assay
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Neutrophils (1.5x106 cells) were incubated for 30 min in a 24-well polystyrene plate. Following a medium change, cells were infected with S. sanguinis at an MOI of 10. Cells treated with 200 nM PMA served as a positive control. After 1 h of incubation at 37°C, cells were suspended with Laemmli gel loading buffer [6.25 mM Tris-HCl (pH 6.8), 4% sodium dodecylsulfate, 10% glycerol, 0.005% bromophenol blue, 50 mM dithiothreitol]. Histone citrullination was detected by immunoblot analysis with a monoclonal antibody against citrullinated histone H3 (rabbit, 1:2000, Abcam, Cambridge, UK). As a loading control, β-actin was detected using an anti-β-actin antibody (rabbit, 1:2000, Cell Signaling, MA, USA). Horseradish peroxidase (HRP)-conjugated antibody against rabbit IgG (1:2000, Cell Signaling) was used as a secondary antibody. Immunoreactive bands were detected using Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, IL, USA).
5
Quantitative Western Blot Analysis
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A 15 μg aliquot of protein per sample was resolved on a denaturing 10% polyacrylamide gel, and proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a semidry blotter by established standard protocols (Figueroa-Romero et al., 2009 (link); Lunn et al., 2009 (link)). Blots were probed overnight at 4°C with the following primary antibodies: rabbit anti-TDP43 (1:1,000; cat#A260, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-histone 2B (H2B) member S (1:500; cat# NB100-56347, Novus Biologicals, Littleton, CO, USA), rabbit anti-GFP (1:1,000; cat#2555, Cell Signaling Technology), and rat anti-α-tubulin (1:5,000; cat#ab6160, Abcam). The next day, blots were rinsed and probed for 50 min at room temperature with appropriate HRP-conjugated secondary antibodies (1:1,000; Santa Cruz Biotechnology or New England Biolabs), then rinsed and visualized by enhanced chemiluminescence with Prime Western Blotting Detection Reagent (Amersham, GE Healthcare, Chicago, IL, USA).
6
Immunoblotting Analysis of Mitochondrial Dynamics
7
Western Blot Analysis of His-Tagged Proteins
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Gels were blotted to nitrocellulose membranes (Protran83; Schleicher & Schuell) for 1 h constantly at 300 mA, blocked for 1 h at room temperature and incubated for 1 h with the anti-His5 horseradish peroxidase-conjugated antibody following the manufacturer's instructions (Penta-His HRP Conjugate Kit, Qiagen). Membranes were washed twice in TBS-Tween-Triton buffer and once in TBS buffer for 10 min each. Detection was followed by incubating with enhanced chemiluminescence Prime Western Blotting Detection Reagent (GE-Healthcare). As a molecular weight standard, Seeblue 2 (Invitrogen) was used. For the purified domains, western blotting intensities were analysed using the integrated density function of ImageJ (https://imagej.nih.gov/ij/index.html ).
8
Quantification of ICAM-1 Expression in Ischemic Brain
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Tissue samples obtained from the reperfused ischemic middle cerebral artery territory and homologous contralateral brain tissue were lysed in NP40 lysis buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 2 mM EDTA) containing protease inhibitor cocktail (Roche, Mannheim, Germany). Equal protein amounts (20 µg) from individual animals were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare Life Science, Little Charfont, U.K.). Non-specific binding was blocked for 1 h at room temperature with 5% skim milk powder (Sigma-Aldrich, Deisenhofen, Germany) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated overnight at 4 °C in goat anti-ICAM-1 (AF796; R&D Systems, Minneapolis, MN, U.S.A.) or rabbit anti-β-actin (4967; Cell Signaling Technology, Danvers, MA, U.S.A.) antibody diluted in TBS-T, followed by incubation for 1 h at room temperature in HRP-conjugated secondary antibody (Santa Cruz Biotechnology) diluted in TBS-T. Signals were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science). ICAM-1 abundance was normalized to β-actin abundance.
9
Western Blot Analysis of VEGFR2 and ASM
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Cells were lysed with NP-40 buffer containing protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were centrifuged at 13,400 rpm at 4 °C and the supernatant was collected. Equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to nitrocellulose membranes (GE Healthcare Life Science). Non-specific binding was blocked with 5% non-fat milk powder (Sigma-Aldrich) dissolved in 0.1% Tween in 0.1 M Tris-buffered saline (TBS-T). Membranes were incubated with rabbit anti-vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) (55B11; Cell Signaling Technology), goat anti-ASM (AF5348, R&D Systems) and rabbit anti-β-actin (4967; Cell Signaling Technology) antibody overnight at 4 °C, rinsed and incubated in peroxidase-conjugated secondary antibodies (Santa Cruz, Heidelberg, Germany) for 1 h at room temperature. Signals were detected by enhanced chemiluminescence using prime Western blotting detection reagent (GE Healthcare Life Science). VEGFR2 and ASM expression were normalized to β-actin abundance.
10
Western Blotting Analysis of Signaling Proteins
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For western blotting, 10 mg of protein was electrophoresed with 4%‒12% PROTEAN precast gels (Bio-Rad) and transferred to a polyvinylidene fluoride blotting membrane (GE Healthcare Life Science). The membrane was incubated with the primary antibody overnight at 4°C, followed by incubation with the secondary antibody conjugated with horseradish peroxidase (1:2,000). Signals were detected and visualized using Amersham ECL (enhanced chemiluminescence) Prime western blotting detection reagent (GE Healthcare). Luminescence was detected using a ChemiDoc MP imaging system (Bio-Rad).
Quantification of the band intensity was performed with Image Lab software version 5.0 (Bio-Rad). Day 3 samples were analyzed. The following primary antibodies were used: anti-phosphorylated-JNK, anti-JNK (1:1,000; Abcam); anti-phosphorylated-ERK, anti-ERK (1:1,000; Cell Signaling Technology); anti-phosphorylated-PI3K, anti-PI3K (1:1,000; Abcam); and anti-GAPDH (1:1,000; Abcam). Amersham ECL anti-rabbit immunoglobulin G (IgG) (GE Healthcare) and Amersham ECL anti-mouse IgG (GE Healthcare) were used as secondary antibodies, each at a dilution of 1:2,000.
Quantification of the band intensity was performed with Image Lab software version 5.0 (Bio-Rad). Day 3 samples were analyzed. The following primary antibodies were used: anti-phosphorylated-JNK, anti-JNK (1:1,000; Abcam); anti-phosphorylated-ERK, anti-ERK (1:1,000; Cell Signaling Technology); anti-phosphorylated-PI3K, anti-PI3K (1:1,000; Abcam); and anti-GAPDH (1:1,000; Abcam). Amersham ECL anti-rabbit immunoglobulin G (IgG) (GE Healthcare) and Amersham ECL anti-mouse IgG (GE Healthcare) were used as secondary antibodies, each at a dilution of 1:2,000.
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