Gravid adult worms were transferred to tubes containing M9 buffer (22 mM KH2PO4, 42 mM Na2HPO4, 86 mM NaCl, and 1 mM MgSO4•7H2O), washed 4 times with M9 containing 0.1% Tween-20 and then resuspended on 1.5X sample buffer (87.5 mM Tris-HCl pH 6.8, 2.5% SDS, 150 mM DTT, 7.5% glycerol, bromophenol blue). After lysing by sonication and boiling, the equivalent of 8–12 worms was loaded onto 4–12% NuPAGE Bis-Tris Gels (Invitrogen). Proteins were then transferred to nitrocellulose membranes, probed with primary antibodies (see Key Resources Table ) and detected using either horseradish (HRP)- conjugated secondary antibodies and WesternBright Sirius (Advansta) chemiluminescent substrate or, for the α-loading control, using alkaline phosphatase (AP)-conjugated secondary antibody and Western Blue® Stabilized Substrate for Alkaline Phosphatase (Promega). Membranes were imaged using a ChemiDoc MP imaging system (BioRad). For immunoblotting of U2OS samples, cells were collected by trypsinization, washed once with 1X PBS, resuspended on 1.5X sample buffer and boiled for 5 minutes before loading onto gels.
Westernbright sirius
Manufactured by Advansta
Sourced in United States, Germany
WesternBright Sirius is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It provides a sensitive and stable signal for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (8 mentions)
Supersignal west femto, by Thermo Fisher Scientific (6 mentions)
Mini protean gel, by Bio-Rad (5 mentions)
Hrp conjugated secondary antibody, by GE Healthcare (5 mentions)
Chemidoc mp system, by Bio-Rad (5 mentions)
Trans blot turbo system, by Bio-Rad (5 mentions)
Protein assay, by Bio-Rad (5 mentions)
Qubit 3 fluorometer, by Thermo Fisher Scientific (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Western blue stabilized substrate for alkaline phosphatase, by Promega (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ecl chemostar, by Intas (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Enhanced chemifluorescence detection system, by Cytiva (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Goat anti rabbit hrp, by Cell Signaling Technology (2 mentions)
Goat anti mouse hrp, by Cell Signaling Technology (2 mentions)
58 protocols using westernbright sirius
1
Immunoblotting Analysis of Gravid Worms
2
Protein Detection and Quantification
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Preparation of whole-cell, cytosolic, and nuclear extracts, determination of protein concentrations, electrophoresis, and Western blotting were performed as previously described [7 (link)]. For the detection of specific proteins, membranes were incubated (4 °C, overnight) with primary antibodies. Following incubation with horseradish peroxidase-coupled secondary antibodies, protein bands were visualised using enhanced chemoluminescence (ECL), SuperSignal West Femto (Thermo Fisher), or WesternBright Sirius (Advansta, Menlo Park, CA, USA) and the Bio-imaging system ECL Chemostar (Intas Science Imaging, Göttingen, Germany). For densitometric analyses, the ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA) was used.
3
Extraction and Detection of Transiently Expressed Proteins
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Transiently expressed LRX8 and RALF4 proteins in tobacco were extracted as follow: 300 mg of plant material were crushed in liquid nitrogen and resuspended in 600 µL of extraction buffer (150 mM Tris pH 7.5; 150 mM NaCl; 10% [vol/vol] glycerol; 10 mM EDTA, 0.5% IGEPAL, cOmplete protease inhibitor [Roche]). Samples were centrifuged at 15,000 rpm for 20 min, and the supernatant was then centrifugated twice more to discard residual pellet debris. A total of 50 µL of input sample was kept at this stage, the rest was incubated for 1 h at 4 °C with anti-myc or anti-HA magnetic beads (µMACS, Milteny Biotech). Immunoprecipitation was performed according to manufacturer’s specifications, using the extraction buffer for column washes. Standard Laemmli buffer (56 (link)) was added to the input and immunoprecipitated samples prior to gel loading. Finally, Western blot analysis was performed with horseradish peroxidase-coupled anti-myc and anti-HA antibodies (MACS, Milteny Biotech) at 1:2,000 dilution. Detection was performed using WesternBright Sirius (Advansta). Images were taken with a LAS500 BlotImager. For Western blot detection of expressed proteins in insect cells (supernatant and pellet fractions) we used anti-His HRP conjugated from Roche in a 1:2,000 dilution.
4
Protein Extraction and Western Blotting
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Total protein extracts from cells were prepared using RIPA buffer (Beyotime Institute of Biotechnology, Nantong, China). Proteins were fractionated in SDS–polyacrylamide gels, transferred to polyvinylidene fluoride (Millipore, Billerica, MA, USA), and western blotting were performed by using the appropriate antibody. Antibody/antigen complexes were detected using ECL (Western Bright Sirius; Advansta, Inc., Menlo Park, CA, USA) and images were acquired using an enhanced chemifluorescence detection system (Amersham Biosciences, Piscataway, NJ, USA) under the room temperature.
For coimmunoprecipitation, Total protein lysates (0.5 mg) were incubated with 4 μg of specific anti-NFBD1 antibody overnight at 4 °C. NFBD1 immunocomplexes were captured with SureBeadsTM Magnetic Beads (Bio-Rad, Hercules, CA, USA) for 1 h at 4 °C. The resulting immunocomplexes were collected by centrifugation, were washed, boiled in SDS sample buffer, loaded on an SDS–polyacrylamide gel. Proteins were analysed by western blotting using standard methods and detected as described above.
For coimmunoprecipitation, Total protein lysates (0.5 mg) were incubated with 4 μg of specific anti-NFBD1 antibody overnight at 4 °C. NFBD1 immunocomplexes were captured with SureBeadsTM Magnetic Beads (Bio-Rad, Hercules, CA, USA) for 1 h at 4 °C. The resulting immunocomplexes were collected by centrifugation, were washed, boiled in SDS sample buffer, loaded on an SDS–polyacrylamide gel. Proteins were analysed by western blotting using standard methods and detected as described above.
5
Immunoblotting Workflow for Protein Detection
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For immunoblotting, cells were cultured in 10 cm plates, harvested at 50–80% confluence and lysed by sonication in RIPA buffer + protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Cell extracts were stored at −80°C until use. Before use, extract concentrations were normalized based on a Protein Assay (Bio-Rad). For every sample, 20–30 μg protein/lane was run on Mini-PROTEAN gels (Bio-Rad), and transferred to PVDF membranes using a TransBlot Turbo system (Bio-Rad). Blocking and antibody incubations were performed in TBS-T + 5% nonfat dry milk. Detection was performed using HRP-conjugated secondary antibodies (GE Healthcare) with WesternBright Sirius (Advansta) or SuperSignal West Femto (Thermo Fisher Scientific) substrates. Membranes were imaged on a ChemiDoc MP system (Bio-Rad).
6
EV Protein Quantification and Analysis
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Protein concentrations of EV were measured, after lysis with 0.2% sodium dodecylsulphate (SDS) (L3771-500G, Sigma, Diegem, Belgium), with the Qubit Protein assay kit (ThermoFisher, Waltham MA, USA) and Qubit fluorometer 3.0 following manufacturer’s instructions. Protein concentrations of cell lysates, obtained in Laemmli lysis buffer (0.125 M Tris–HCl [pH 6.8], 10% glycerol, 2.3% SDS), were determined using the Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA, USA). For protein analysis, samples were dissolved in reducing sample buffer (0.5 M Tris-HCl [pH 6.8], 40% glycerol, 9.2% SDS, 3% 2-mercaptoethanol, 0.005% bromophenol blue) and boiled at 95 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked for 30 min in blocking buffer (5% non-fat milk in PBS with 0.5% Tween-20) and incubated overnight at 4 °C with primary antibodies. Secondary antibodies were added for 60 min at room temperature after extensive washing with blocking buffer. After final washing, chemiluminescence substrate (WesternBright Sirius, Advansta, Menlo Park, CA, USA) was added and imaging was performed using Proxima 2850 Imager (IsoGen Life Sciences, De Meern, The Netherlands). Quantification of signal intensity was performed using ImageJ software.
7
Western Blot Analysis of Retinal Proteins
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Preparation of retinal extracts and Western blot was performed as previously described69 (link). In order to avoid biased matched comparisons, samples from both retinas of each animal were loaded, side by side in the gel, assuming separated membranes for control and diabetic animals. Nevertheless, membranes were developed at the same time and comparisons were made to the β-actin ratio. The membranes were incubated with the antibodies described in Table 2 . Immunolabeling was detected using WesternBright Sirius™ (Advansta, Menlo Park, CA, USA) or with ECF™ (GE Healthcare Amersham™, Little Chalfont, UK), in accordance with the manufacturer’s instructions.
8
SDS-PAGE and Immunoblotting Analysis
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SDS-PAGE and immunoblotting analysis were performed as previously described (58 (link)). In brief, denatured cell extracts from 200,000 cells were resolved on either gradient Tris–Glycine acrylamide gels (Bio-Rad) or home-made 10 to 16% Tris–Glycine acrylamide gels. Proteins were transferred to a polyvinylidene difluoride membrane using a wet transfer protocol and blocked with 5% skimmed milk for 1 h. Membranes were incubated with the appropriate primary antibody overnight at 4 °C, washed with PBS with Tween-20, and then incubated with a secondary antibody for 1 h at RT. The membranes were visualized using either Clarity (catalog no.: 1705061; Bio-Rad) or WesternBright Sirius (catalog no.: K-12043-D10; Advansta) enhanced chemiluminescence solutions and a ChemiDoc Imaging System equipped with the ImageLab software (Bio-Rad Laboratories).
9
Western Blotting of ADAR1 in Transfected Cells
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For western blotting HeK293 cells or MEFs were grown in DMEM/FBS/Pen-Strep. To induce the production of ADAR1, cells were transfected with FLAG-tagged ADAR1p150 fused to T2A-GFP in pcDNA 3.1.
For induction of endogenous ADAR1, cells were treated over night with mouse IFN-α (Hycult Biotech, Uden, Netherlands) to 1000 units/ml, final concentration. After over-night expression, cells were washed and directly lysed in 2× SDS sample buffer (33 (link)). Cell lysates were sonicated to shear DNA, denatured at 98°C and loaded onto a 7% SDS protein gel. After blotting onto nitrocellulose membrane, proteins were detected with an anti FLAG-antibody, or with mouse mAb 15.8.6 (1:300, Santa Cruz Biotechnology, order nr. sc-73408) directed against ADAR1. Blots were further developed with secondary HRP-coupled antibodies (1:5000, Pierce, 31444) and detected via chemiluminescence (WesternBright Sirius, advansta, Menlo Park, CA, USA). Alternatively, blots were detected with a rabbit anti ADAR1-p150 antibody (1:1000, SynapticSystems, order nr. 293003) and goat anti-Rabbit IgG1 HRP conjugate (1:5000, Cell Signaling Technologies, order nr. 7074).
For induction of endogenous ADAR1, cells were treated over night with mouse IFN-
10
Western Blot Analysis of PLS3 Expression
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U2OS cells transfected with mEmerald-PLS3 constructs were harvested and cell lysates were prepared in the reducing SDS-PAGE sample buffer. Samples were subjected to PAGE and transferred to nitrocellulose. Membranes were blocked in PBS containing 0.1% Tween-20 and 5% nonfat dry milk for 1 h at room temperature and incubated with anti-PLS3 antibody (Sigma SAB2700266; 1:1 000) in the blocking buffer overnight at 4 °C. Following three washes with PBS containing 0.1% Tween-20, membranes were incubated with anti-rabbit antibody conjugated with horseradish peroxidase (HRP; Sigma A0545; 1:10 000) for 1 h at room temperature. Signal was detected using chemiluminescent HRP substrate WesternBright Sirius (Advansta) in an Omega Lum G imager (Aplegen).
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