Cells were gently washed in PBS and then lysed using lysis buffer (1% NP-40, 50 mM Tris–HCl pH 7.5, 150 mM NaCl, complete protease inhibitor tablet, EDTA-free (Roche), 1 μM sodium orthovanadate, 1 mM sodium fluoride, and 0.1% β-mercaptoethanol). Lysed cells were scraped and transferred into a 1.5-ml microcentrifuge tube. Samples were incubated at 4°C for 20 min and then centrifuged for 15 min at 17,000x g at 4°C. Proteins (50 μg) were resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Alternatively, the cleared cell lysates were incubated with anti-Flag M2 affinity agarose beads (Sigma-Aldrich), overnight at 4°C. Then the beads were washed three times with the lysis buffer then resuspended with sample buffer and then resolved on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Western blots were probed with the following antibodies: anti-Flag (M2) (Sigma-Aldrich), anti-c-NRAS (F155-277, Millipore), anti-NRAS (sc-519, Santa Cruz), anti-c-CBL (2747, Cell signaling), and anti-αtubulin (Millipore).
Nitrocellulose membrane
Manufactured by Bio-Rad
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Nitrocellulose membranes are a type of laboratory equipment designed for use in protein detection and analysis techniques. These membranes serve as a support matrix for the immobilization of proteins, enabling various downstream applications such as Western blotting, dot blotting, and immunodetection.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (236 mentions)
Ripa buffer, by Thermo Fisher Scientific (188 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (181 mentions)
Odyssey infrared imaging system, by LI COR (164 mentions)
Protease inhibitor cocktail, by Merck Group (136 mentions)
Image lab software, by Bio-Rad (126 mentions)
Protease inhibitor cocktail, by Roche (120 mentions)
β actin, by Merck Group (114 mentions)
Chemidoc mp imaging system, by Bio-Rad (113 mentions)
Clarity western ecl substrate, by Bio-Rad (94 mentions)
β actin, by Cell Signaling Technology (89 mentions)
Ripa buffer, by Merck Group (86 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (84 mentions)
Bovine serum albumin (bsa), by Merck Group (82 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (74 mentions)
Bradford assay, by Bio-Rad (73 mentions)
Protease inhibitor, by Roche (73 mentions)
Chemidoc imaging system, by Bio-Rad (71 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (69 mentions)
Odyssey blocking buffer, by LI COR (68 mentions)
5 040 protocols using nitrocellulose membrane
1
Western Blot Analysis of NRAS and CBL Proteins
2
Western Blot Protein Quantification and Detection
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Cellular lysates were obtained solubilising about 3 × 10 6 of cells in 0.1 ml of lysis buffer (1% Triton-X-100, 0.5 mM EDTA, 0.6 mM PMSF, 100 μl/ml of protease inhibitor cocktail dissolved in phosphate buffered saline at pH 7.4), sonicated and centrifuged at 10000 ×g. The protein content of the supernatant (cell lysate) was determined according to Bradford (Bradford 1976 ) and 60 μg of proteins were separated by 12% SDS-PAGE. Gels were transferred onto nitrocellulose membrane (BioRad) for 1 h at 100 V. The nitrocellulose membranes were incubated overnight at 4 °C with the indicated antibody. Primary antibodies were visualized using horseradish peroxidase-conjugated secondary antibodies (1:2000) . The chemiluminescence signals were revealed using an ECL Western blotting kit (Amersham Bioscience, Buckinghamshire, UK) and measured with Gel Logic 1500 Imaging System, Kodak.
3
Immunoblotting of Nuclease-Treated PEARL Extracts
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Nuclease-treated PEARL extracts were separated on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad) for Western blot analysis. The membranes were blocked in 0.5% bovine serum albumin–Tris-buffered saline with Tween (BSA-TBST) for 30 min. Primary antibodies were diluted in 0.5% BSA-TBST as follows: anti-HSP-70 (Cell Signaling Technology 4872), 1:1,000; anti-LANA/ORF73 (Advanced Biotechnologies 13–210-100), 1:3,000; anti-GFP (Invitrogen A11122), 1:3,000; and anti-NS2B (GeneTex GTX133308), 1:1,000. The membranes were incubated with primary antibodies for 30 min at room temperature. Following primary-antibody incubation, the membranes were washed with TBST 3 times before the addition of horseradish peroxidase (HRP)-conjugated secondary antibodies. The membranes were incubated for 30 min with secondary antibody diluted 1:3,000 in 0.5% BSA-TBST. Immunoreactivity was detected with Radiance Plus HRP substrate (Azure Biosystems). All images were captured with an Azure Biosystems C300 gel imaging system. Image postprocessing was carried out in Photoshop CC (Adobe) using automatic contrast. For dot blot-based immunodetection, nitrocellulose membranes (Bio-Rad) were spotted with 1 μl of PEARL extract and allowed to dry completely at room temperature for 30 min. For the remainder of the procedure, membranes were processed and imaged as described for Western blotting.
4
Western Blot Analysis of Mitochondrial Proteins
5
Western Blot Analysis of Macrophage Signaling
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After treatment with compounds and LPS, RAW264.7 macrophages were washed with phosphate buffered saline and harvested with cell lysis. After being centrifuged at 12,000 rpm at 4°C for 10 minutes, the protein concentration was determined. After the sample loading buffer was added, protein samples were electrophoresed and then transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). Each membrane was blocked for 1.5 hours at room temperature and incubated with specific primary antibodies against p-ERK (1:300), ERK (1:300), p-P38 (1:1,000), P38 (1:1,000), p-JNK (anti-p-Jun N-terminal kinase) (1:1,000), JNK (1:1,000), IkBα (1:300), and GAPDH (1:1,000) at 4°C overnight. Following three washes with Tris-buffered saline, the nitrocellulose membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour, and visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The amounts of the proteins were analyzed using ImageJ analysis software version 1.38e (National Institutes of Health, Bethesda, MD, USA) and normalized to their respective controls.
6
Recombinant Protein Immunoreactivity Profiling
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Purified recombinant proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and stained with Coomassie blue R-250 (Bio-Rad) or with Ponceau S (ThermoFisher, Waltham, MA, USA) after electrotransformation onto nitrocellulose membranes (Bio-Rad). For Western blot analysis, recombinant proteins were electrotransferred onto nitrocellulose membranes, which were immunoblotted with pooled 300 H. pylori-positive sera verified previously at a 1:200 dilution [28 (link)] and an alkaline phosphatase-conjugated goat anti-human IgG (H + L) at a 1:500 dilution (Bio-Rad). Specific reactions were detected using an AP-conjugated substrate kit (Bio-Rad). The reactivity of immunoblot was compared and graded using the mean gray value function of the ImageJ software (1.53e version) after background compensation. The relative antigenicity values are the ratio of Western blot reactivity to the protein staining levels in SDS-PAGE (loading control).
7
Hippocampal Expression of ApoE, Tau, and Related Proteins
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Full‐length ApoE, ApoE fragments, and Tau5, AT8, and PHF1 expressions in the hippocampus was detected using western blotting. Using a 4–20% double TIS polyacrylamide gel (Cat#M00655, Gen Script Biotech Corp., Nanjing, China) or a standard XT 4–12% double TIS gel (Bio‐Rad, USA), the samples were separated from SDS‐PAGE and transferred to nitrocellulose membranes (Bio‐Rad, USA). The membranes were then blocked with 5% skimmed milk powder and identified using the following primary antibodies: Tau5 (1:1000, Cat#ABN454, Millipore, USA), full‐length ApoE and ApoE fragments (1:4000, Cat#178479, Calbiochem, Germany), PHF1 (1:1000, Cat#3Ab184951, Abcam, UK), and AT8 (1:2000, Cat#MN1020, Thermo Fisher Scientific, USA). After washing at 4°C overnight, the membranes were incubated at 37°C for 1 h with goat anti‐mouse antibody (1:5000, Cat#31430, Invitrogen, USA). The nitrocellulose membranes were then scanned and photographed using Bio‐Rad image analysis equipment after being washed in TBST and dripped with ECL chemiluminescent liquid (Cat#34577, Invitrogen, USA). The ratio of the integral optical density of the target band to that of the β‐actin band represents the level of expression of the target protein. A 100% change refers to control or sevoflurane levels for comparison between the experimental settings.
8
Protein Extraction and Western Blotting
9
Dot-Blot and Western Blot Analysis
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For dot-blot assays, nitrocellulose membranes placed in a blotter device (BioRad, Hercules, United States) were directly coated with proteins from cell culture SN at 150 µL/well. The membranes were blocked with tris-buffered saline, pH 7.6, containing 5 % powdered non-fat milk and 0.05 % Tween 20 and developed with mouse anti-His-C Ab (Invitrogen) diluted 1:5.000, goat anti-mouse IgG alkaline phosphatase-conjugate diluted 1:500 (Sigma-Aldrich), and alkaline phosphatase substrate (5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium, Sigma Aldrich). For Western blot analysis, samples of SN or cell lysate from similar volumes of cultures of High-five cells infected with AcBac-pFast-cont, AcBacΔCC-pFast-cont, AcBac-sccaIL-12, AcBacΔCC-sccaIL-12, AcBac-sccaIL-12opt or AcBacΔCC-sccaIL-12opt baculovirus contructs at a MOI 5 for 48 h were fractioned by polyacrylamide gel electrophoresis with dodecyl sodium sulfate (SDS–PAGE) and then transferred to nitrocellulose membranes (BioRad). Detection of his-tagged protein was performed as described in the dot-blot assay.
10
Quantification of Polyubiquitinated Substrates
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A total of 10 OD600 units of yeast grown to log phase in selection media was harvested and lysed mechanically using zirconium beads (BioSpec Products). Cell lysates were precipitated and prepared with TCA precipitation as above. Small equal volumes of each lysate sample were resolved on a 4–15% gradient SDS-PAGE gel (Biorad) and transferred onto a nitrocellulose membrane (Biorad) for quantification and normalization of HA-tagged substrates in each sample with immunoblot. Normalized volumes of proteins were immunoprecipitated with the anti-HA affinity matrix (Roche). Proteins were resolved on a 4–15% gradient SDS-PAGE gel (Biorad) and transferred onto a nitrocellulose membrane (Biorad), autoclaved and immunoblotted for polyubiquitinated substrates. The membrane was scanned with the Odyssey Li-Cor Imaging System and the bands were quantified with ImageJ (Schindelin et al. 2012 (link)).
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