Protein extracts were prepared using radioimmunoprecipitation assay lysis buffer (Beyotime, P0013B). Proteins were separated through a 4 to 12% SDS–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membrane (Roche, 03010040001). After blocked by blocking reagent (Roche, 11096176001) for 1 hour, the membrane was incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation. Images were captured using a ProteinSimple FluorChem system. Filia antibody was from the previous study (14 (link)). Other commercial antibody information was listed in table S2.
Nitrocellulose membrane
Manufactured by Roche
Sourced in Germany, Switzerland, United States, United Kingdom
Nitrocellulose membrane is a semi-permeable material that is commonly used in various laboratory applications. It is made from cellulose that has been treated with nitric acid, resulting in a porous structure that allows for the transfer and immobilization of biomolecules such as proteins and nucleic acids. The membrane's primary function is to serve as a support matrix for analytical techniques like Western blotting, dot blotting, and immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (3 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (2 mentions)
Anti cyclin d1, by Santa Cruz Biotechnology (2 mentions)
Anti cyclin b1, by Santa Cruz Biotechnology (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Anti tubulin, by Merck Group (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pcr dig probe synthesis kit, by Roche (2 mentions)
Anti flag, by Merck Group (2 mentions)
Blocking reagent, by Roche (1 mentions)
Vision work ls analysis software, by Analytik Jena (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Ab28146, by Abcam (1 mentions)
Horseradish peroxidase, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Bcip nbt substrate, by Merck Group (1 mentions)
51 protocols using nitrocellulose membrane
1
Western Blot Analysis of Filia Protein
2
Quantification of P450 Protein Levels
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P450 protein expression levels were determined by Western blotting [25 (link)] with beta-actin (ab8227; Abcam, USA) as a housekeeping control. Briefly, total protein was extracted from 30 mg of fresh liver tissues using RIPA buffer (50-mM Tris pH 8, 150-mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) supplemented with a phosphatase inhibitor cocktail (Roche, Canada). Next, aliquots containing 100 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Roche, Canada) using a Pierce Fast Semi-Dry Blotter (Pierce, USA). The membrane was blocked with 1% skimmed milk for 2 h, washed with TBST buffer (1.5-M NaCl, 0.5-M Tris, pH 7.5) three times, and incubated with an anti-cytochrome P450 2E1 antibody (ab28146; Abcam, USA). Next, the membrane was incubated with a secondary antibody conjugated with horseradish peroxidase (Abcam, USA). The chemiluminescence signals (Super Signal West Pico; Pierce, USA) were developed using a Chemi Doc UVP machine (UVP, USA). The density results were analyzed using Vision Work LS Analysis software (UVP, USA).
3
Protein Analysis by SDS-PAGE and Western Blot
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Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 10% polyacrylamide gel. Proteins were visualized by staining of the gel by Coomassie Brilliant Blue R-250. For western blot analysis, bacterial lysates or purified proteins from different bacterial clones were subjected to SDS-PAGE and transferred to nitrocellulose membrane (Roche, Germany). The membrane was then incubated with primary mouse monoclonal anti-histidine tag-HRP (Roche, Germany) and/or polyclonal anti-chitinase (raised in rabbit). Goat anti-rabbit HRP (Roche, Germany) was used as the secondary antibody conjugate for polyclonal antibody, and the blot was developed with diaminobenzidine tetrahydrochloride (DAB) substrate (Sigma, USA). Equal amount of proteins were used in all experiments.
4
Western Blot Protein Analysis Protocol
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The cells were washed twice with ice-cold PBS buffer, and total protein lysates were prepared using radioimmunoprecipitation assay buffer supplemented with protease inhibitors (Beyotime, Nantong, China). Aliquots of protein lysates (30 μg) were electrophoresed on 8% (for ARID1A) or 10% sodium dodecyl sulfate-polyacrylamide gels (for other proteins), and transferred to a nitrocellulose membrane (Roche, Switzerland). Next, the membranes were incubated with primary antibodies purchased from Cell Signaling Technology (Danvers, MA, USA) overnight at 4°C, then with the appropriate horseradish peroxidase-conjugated secondary antibody. Immunoreactivity signals were visualized using chemiluminescence detection reagent and imaged.
5
Western Blot Analysis of SHBsAg
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Leaf fragment or lyophilised tissue, approx. 50–60 mg in weight, were ground in 300 μL of PBS with 0.5% (v/v) Tween 20, then Laemmli buffer with 100 mM DTT was added and samples were incubated at 65°C for 15 min. Samples were run on 12% polyacrylamide gel with 0.1% (w/v) SDS and then blotted onto a nitrocellulose membrane (Roche). The blot was blocked with 3% (w/v) BSA in TBS and then incubated with the TBS-diluted rabbit polyclonal anti-SHBsAg antibody (Cat. No. B65811R, Meridian Life Science). The AP-conjugated goat polyclonal anti-rabbit whole-molecule antibody (Sigma) followed. The molecular weight of protein bands, visualized after a reaction with the BCIP/NBT substrate (Sigma), were estimated using a protein size marker (MBI Fermentas).
6
Western Blot Analysis of Cell Signaling
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Total protein was extracted using a radioimmunoprecipitation assay (RIPA) lysis buffer (Wolsen, Xi’an, P.R. China) from cells or tissue samples, separated in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and electrophoretically transferred to nitrocellulose membrane (Roche, Basel, Switzerland). Then the membrane was incubated with primary antibodies overnight at 4°C. The primary monoclonal antibodies included rabbit monoclonal anti-β-catenin (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit monoclonal anti-c-Myc (1:1,000; Santa Cruz Biotechnology), mouse monoclonal anti-cyclin D1 (1:1,000; Santa Cruz Biotechnology), and mouse monoclonal anti-GAPDH (1:2,000; Santa Cruz Biotechnology). The membranes were incubated with enhanced chemiluminescence (ECL; Pierce, Rockford, IL, USA) for chemiluminescence detection. The blots were scanned, and the band density was measured with Quantity One imaging software.
7
Immunoblot Analysis of CTX Protein
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For immunoblot analysis, proteins were electro-phoretically transferred onto a nitrocellulose membrane (Roche, Germany) using transfer buffer containing 39 mM glycine, 48 mM Tris-base, 0.037% SDS, and 20% methanol. The membrane was blocked with 5% skimmed milk in PBST (13 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4 and 0.05% (v/v) Tween-20) overnight at 4 °C. The nitrocellulose membrane was washed three times with PBST and incubated for 1 hr with anti-CTX (Sigma, Germany) at 1:5000 dilution. Following washing the membrane with PBST, anti-rabbit IgG conjugate (Dako, Denmark) was added and incubated for 1 hr at 37 °C. Finally, detection was carried out using an HRP staining solution containing DAB (Sigma, Germany). Chromogenic reaction was halted by rinsing the membrane twice in distilled water.
8
Protein Extraction and Western Blot Analysis
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Protein extracts were prepared from primary cultured DRG neurons. The cultured DRG neurons were washed twice with PBS and lysed with RIPA buffer (Thermo Fisher Scientific) with protease inhibitor (Roche Diagnostics) and phosphatase inhibitor (Roche Diagnostics) at 4°C for 30 min to extract proteins. The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). Equal quantities of protein were electrophoresed on 10% SDS-PAGE and transferred onto a nitrocellulose membrane (Roche Diagnostics). The membranes were incubated with the primary antibody overnight at 4°C after blocking with 5% milk dissolved in TBST buffer for another 2 hr at room temperature (25℃). The membranes were washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody for 1.5 hr at room temperature, followed by chemiluminescent detection after incubation with ECL substrate (Thermo Fisher Scientific). The blots were probed with the candidate antibodies shown in Key Resources Table. ImageJ software was used to quantify the results of the western blot.
9
Protein Expression Analysis by Immunoblotting
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Immunoblotting was performed as previously reported [16 (link)]. In brief, total proteins were extracted from the cultured cells with radioimmunoprecipitation (RIPA) assay buffer (Cell Signaling Technology, Boston, MA). Samples containing 30–35μg of total protein were loaded onto 8–12% SDS polyacrylamide gel electrophoresis (PAGE), transferred onto a nitrocellulose membrane (Roche), and probed with primary antibodies. Anti-bcl-2, anti-NCL, anti-Akt1, anti-cyclin A1, anti-cyclin B1, and anti-cyclin D1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin antibody (AC-40) was purchased from Sigma-Alderich; Anti-p53 monoclonal antibody (clone Y5) was purchased from abcam (Cambridge, MA). Following incubation with HRP-conjugated goat anti-rabbit, or goat anti-mouse secondary antibodies (ZSGB-BIO), protein bands were visualized by an ECL plus chemiluminescence kit (Beyotime, Haimen, China).
10
Western Blot Analysis of Cell Signaling
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Stimulated cells were rinsed with ice-cold PBS and lysed using lysis buffer (iNtRon Biotech, Seoul, South Korea) for 1 h. Total cell lysates were centrifuged at 12,000×g at 4 °C for 10 min to obtain supernatants. After bicinchoninic acid (BCA, Sigma) protein quantification assay, the supernatant was mixed with 2× sample buffer, boiled at 95 °C for 5 min, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane (Roche Diagnostics, IL, US). These membranes were blocked with 5% skim milk in PBS-Tween-20 (PBST) for 1 h at room temperature followed by overnight incubation with anti-phospho-JNK, anti-p38, and anti-ERK antibodies at 4 °C. After washing three times with PBST, these membranes were incubated with secondary antibodies for 1 h at room temperature followed by three times of washes with PBST. The protein-antibody complexes were visualized with ECL Western blotting Luminol Reagent (Santa Cruz Biotech, CA, USA). Images were recorded with an LAS-4000 image reader (Fujifilm Life Sciences, Tokyo, Japan).
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