The protein samples were mixed with 6 × SDS-PAGE Loading Buffer (Beyotime, Shanghai, China), boiled at 90°C for 5 min and separated on 10% SurePAGE Bis-Tris gels (GenScript, Nanjing, China). Separated proteins were then transferred to nitrocellulose membranes (NC), and the membranes were blocked with PBS containing 5% non-fat milk at 4°C overnight. The membranes were washed three times with PBST, then incubated with the various antibodies for 1 h at 37°C. After washing, HRP-conjugated secondary antibody was added and incubated for 1 h at 37°C. The membranes were washed again and the protein bands were detected with enhanced chemiluminescence (ECL) kit (Thermo, USA).
Surepage bis tris gel
Manufactured by GenScript
Sourced in China, United States
SurePAGE Bis-Tris gels are electrophoresis gels designed for the separation and analysis of proteins. They utilize a Bis-Tris buffer system to provide consistent and reliable protein separation.
Automatically generated - may contain errors
Lab products found in correlation
Dounce tissue grinder, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Mei5 Biotechnology (2 mentions)
Chemiscope 6300, by Clinx (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Ab6721, by Abcam (1 mentions)
Bc3710, by Solarbio (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab13556, by Abcam (1 mentions)
Ab116027, by Abcam (1 mentions)
Pageruler, by Thermo Fisher Scientific (1 mentions)
Amersham imager 680, by GE Healthcare (1 mentions)
Ni charged magbeads, by GenScript (1 mentions)
Bolt lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Maclin Biochemical Technology (1 mentions)
Spectra broad multicolor high range protein ladder standard, by GenScript (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab179484, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Gtx132346, by GeneTex (1 mentions)
16 protocols using surepage bis tris gel
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Cell Signaling Proteins
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Whole cell extracts were harvested according to the instruction (BC3710, Solarbio). Extract concentration was measured by BCA kit (SK1070, Coolaber, Beijing) on microplate reader (M5, Molecular Devices). Cell extracts were separated on 4%–20% SurePAGE Bis-Tris gels (Genscript) and then transferred to PVDF membranes (Millipore). Membranes with proteins were incubated with primary antibodies to be tested at 4°C overnight, then were incubated with fluorescence or HRP conjugated secondary antibodies for 1 h at room temperature. Odyssey SA system was used to detect His tagged MscL, and chemiluminescence was examined by ECL reagent (SL1350, Coolaber, Beijing). The densitometry was determined using Bio-Rad ChemiDoc Touch imaging system (1708371, Bio-Rad). Primary antibodies are as follows: Caspase-3 (9664T, cell signaling technology-CST), Caspase-3 (9746T, CST), Caspase-9 (9502T, CST), Ubiquitin (#58395, CST), Cytochrome-C (#11940, CST), LC3A/B (#12741, CST), TLR4 (ab13556, abcam). Secondary antibodies are as follows: anti-rabbit (8889S, CST), anti-rabbit (ab6721, abcam).
3
Western Blotting of Recombinant Proteins
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Protein preparations for Western blotting from transfected HEK293S cells and tissues are described in the Supplemental Information . Protein samples (1–100 µg, Table S6 ) were loaded alongside the Benchmark (Cat# 10748010, Life Technologies, Carlsbad, CA, USA), PageRuler (Cat# 26616, Thermofisher, Waltham, MA, USA) or ab116027 (Abcam, Cambridge, UK) protein ladders onto 4–12% SurePage Bis-Tris gels (GenScript, Piscataway, NJ, USA). Western blotting was performed as previously described [43 (link)]. Under some conditions, 2-(N-morpholino)ethanesulfonic acid (MES) running buffer was used to aid in the separation of lower molecular weight bands. Figure legends provide detailed information about the conditions used.
4
Purification of Histidine-Tagged Proteins
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Following protein expression, cells were resuspended in Buffer A (20 mM Tris-HCl, 500 mM NaCl, 30 mM Imidazole, pH 8.0) and lysed by sonication. Insoluble material was removed by centrifugation and the soluble lysate was mixed with Ni-charged MagBeads (Genscript, Piscataway, NJ, USA) preequilibrated in Buffer A, washed with Buffer A, and eluted with Buffer B (20 mM Tris-HCl, 500 mM NaCl, 300 mM Imidazole, pH 8.0). Samples were mixed with 4X Bolt LDS Sample Buffer (Invitrogen, Waltham, MA, USA) containing 4% β-mercaptoethanol, heated to 95 °C for 5 min, separated using 8–16% Sure-PAGE Bis-Tris gels (Genscript) with MOPS running buffer, stained by Coomassie, and visualized using an Amersham Imager 680 (GE Healthcare).
5
Western Blotting Methodology for Protein Analysis
6
Fish Skin Preparation and Characterization
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Skins (tilapia, grass carp, and sea perch) were provided by Guangxi Luchi Agriculture Co., Ltd. (Nanning, China); fresh fish skins were frozen under −18 °C, and then transported to the laboratory in refrigerated trucks. The fish skin was manually stripped of scales and muscle fragments using a knife, thoroughly washed in tap water, frozen, and stored at −20 °C before use. Other materials used include trypsin (250 U/mg) (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai, China), 10% SurePAGE Bis-Tris gels (GenScript, Nanjing, China), and SpectraTM Broad Multicolor High Range Protein Ladder Standard (10–180 kDa, GenScript, Nanjing, China).
7
Western Blot Analysis of Protein Expression
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Western blotting analysis was carried out, as previously described (Chen et al., 2021 (link)). Briefly, cells were collected and dissolved within the Lysis Buffer (cat. no. P0013B, Beyotime) containing 1 mM PMSF (cat. no. ST506, Beyotime). The protein concentration was detected through the use of an enhanced BCA Protein Assay Kit (cat. no. P0010, Beyotime). Next, equivalent amounts of protein were separated on SurePAGE Bis-Tris gels (GenScript) for electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were blocked via a blocking buffer (cat. no. P0252, Beyotime) for 1 h, and then cultured with primary antibodies overnight. After incubation with secondary antibodies at appropriate concentration for an hour at room temperature, the protein bands on the membrane were eventually identified using a Hypersensitivity Chemiluminescence Kit (cat. no. G2020, Servicebio). The band intensity was quantified using the ImageJ software (National Institutes of Health).
8
Western Blot Analysis of Protein Samples
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Cell samples were washed with PBS 3 times and collected; brain tissues were ground 20–30 times using Dounce Tissue Grinders (Thermo Fisher, K8853000002). Then the samples were lysed in RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA pH 8.0, 1 mM EGTA pH 8.0, 0.1% SDS, 0.5% DOC, and 1% Triton X-100) with Protease inhibitor cocktail (Mei5bio, MF182-plus-10), and sonicated 6 times. The protein in the samples was separated by SDS-PAGE electrophoresis using SurePAGE™ Bis-Tris Gels (GenScript, M00652), and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 1 h and incubated with the corresponding primary antibodies in 3% BSA at 4 °C overnight. The next day, the membranes were washed three times with 1× PBS and incubated with HRP-conjugated secondary antibodies in 5% skim milk at room temperature for 1 h. The signals were developed with ECL solution (Millipore, WBKLS0500) after washing three times in 1 X PBS. The images were acquired digitally using Clinx ChemiScope 6300.
9
Western Blotting of Stress Proteins
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Cells were treated as indicated and Western blotted as previously described (Liu et al., 2015 (link)). Briefly, the cells were lysed and assessed using 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SurePAGE, Bis-Tris gels, Genscript, Nanjing, China). Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States) and probed with primary antibodies against HSPA6 (sc-374589, Santa Cruz Biotechnology), HSPA1A (PA534772, Invitrogen), HSPA8 (PA5-27337, Invitrogen), HSPB1 (PA1017, Invitrogen), EV71 VP1 (GTX132339, GeneTex), CVA6 VP1 (GTX132346, GeneTex), GAPDH (ab9485, Abcam) or α-Tubulin (ab179484, Abcam). Protein bands were visualized with Pierce™ ECL Western blotting substrate (Thermo Scientific, Rockford, IL, United States). Protein levels were determined by scanning the bands’ intensities and analyzed using Quantity One software (Bio-Rad, Hercules, CA, United States).
10
Western Blot Protein Analysis Protocol
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Cell samples were washed with phosphate-buffered saline (PBS) three times and collected; brain tissues were ground 20 to 30 times using Dounce Tissue Grinders (Thermo Fisher Scientific, K8853000002). The samples were lysed in radioimmunoprecipitation assay buffer [50 mM tris (pH 8.0), 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.1% SDS, 0.5% DOC, and 1% Triton X-100] with a protease inhibitor cocktail (Mei5bio, MF182-plus-10), and sonicated six times. The samples were SurePAGE Bis-Tris gels (GenScript, M00652) for electrophoresis and transferred to a nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 1 hour and incubated with the corresponding primary antibodies in 3% bovine serum albumin (BSA) at 4°C overnight. The next day, the membranes were washed three times in 1× PBS and incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies in 5% skim milk at room temperature for 1 hour. The signals were developed with ECL solution (Millipore, WBKLS0500) after washing three times in 1× PBS. The images were acquired digitally using Clinx ChemiScope 6300.
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