In the analysis of protein expression, cell lysates were homogenized in a RIPA buffer (25 mM Tris, pH 7.4 0.15 M NaCl 0.1% Tween 20) with 1:100 protease inhibitor (78442, Thermo Scientific). The lysates were denatured by boiling, separated on a 4 to 15% SDS/polyacrylamide gel electrophoresis gel (567–1094, Bio-Rad), and transferred to a nitrocellulose membrane (1704159, Bio-Rad). The membrane was blocked using TBS-T with 5% BSA for 1 hour at room temperature and incubated using p-SMAD3 (ab52903, Abcam), type I collagen (1310–30, Southern Biotech), and GAPDH (5174, Cell Signaling). The blot was further washed in TBS-T and incubated at room temperature with its corresponding secondary antibody for 30 minutes. Following another wash, the blot was incubated with ECL (32106, Thermo Scientific) and visualized with the ChemiDox XRS+ image-forming system.
Sds polyacrylamide gel electrophoresis gel
Manufactured by Bio-Rad
Sourced in Canada, United States
SDS–polyacrylamide gel electrophoresis (SDS-PAGE) is a laboratory technique used to separate proteins based on their molecular weight. The gel is made of polyacrylamide, and the proteins are denatured and coated with the anionic detergent SDS, which gives them a uniform negative charge. The proteins are then subjected to an electric field, causing them to migrate through the gel at a rate inversely proportional to their molecular weight.
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Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
P smad3, by Abcam (1 mentions)
Chemidox xrs, by Thermo Fisher Scientific (1 mentions)
Type 1 collagen, by Southern Biotech (1 mentions)
Anti fsp 1, by Merck Group (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti α sma, by Abcam (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti pdgf aa, by Merck Group (1 mentions)
Anti pdgf bb, by Merck Group (1 mentions)
Horseradish peroxidase conjugated antibodies specific for either rabbit or mouse igg, by Bio-Rad (1 mentions)
Anti vegfr2, by Cell Signaling Technology (1 mentions)
Anti pdgfrα, by Cell Signaling Technology (1 mentions)
Anti snail, by Cell Signaling Technology (1 mentions)
Anti nf κb, by Cell Signaling Technology (1 mentions)
16 protocols using sds polyacrylamide gel electrophoresis gel
1
Protein Expression Analysis Protocol
2
Protein Extraction and Western Blotting
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Cells were lysed with a NP-40 lysis buffer containing protease inhibitor cocktail (Roche, 11697498001), followed by measurement of total protein concentration. A total 20 μg protein of the lysates was resolved by 4–15% precast SDS-polyacrylamide gel electrophoresis gel (Bio-Rad). After transfer, polyvinylidene fluoride membranes were blotted with anti-GAPDH (Cell Signaling, 5174), anti-FSP-1 (Millipore, 07-2274, Abcam, ab27957, and Abnova H00006275-M01), anti-VEGFR-2 (Cell Signaling, 9698), anti-N-cadherin (Cell Signaling, 13116), anti-α-SMA (Abcam, ab5694), anti-NF-κB (Cell Signaling, 8242), anti-Snail (Cell Signaling, 3879s), anti-Erg (Cell Signaling, 97249), anti-Slug (Cell Signaling, 9585s), anti-PDGFR-α (Cell Signaling, 3164), anti-PDGFR-β (Cell Signaling, 3169), anti-PDGF-AA (Millipore, 07-1436), and anti-PDGF-BB (Millipore, 07-1437) antibodies at 1:1000 dilution. Proteins were detected with horseradish peroxidase-conjugated antibodies specific for either rabbit or mouse IgG (Bio-Rad), followed by ECL development (GE Healthcare, RPN2232). Uncropped scans of all immunoblots are provided in Supplementary Fig. 9 .
3
Western Blot Protein Detection Protocol
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After treatment, cells were washed with cold phosphate-buffered saline (PBS) and then lysed with cold NP-40 lysis buffer (#BP-119, Boston BioProducts) supplemented with protease inhibitor (#A32965, Thermo Fisher Scientific) and phosphatase inhibitors (#P5726 and #P0044, Phosphatase Inhibitor Cocktail 2,3 from Sigma-Aldrich). Cells were scraped into tubes and centrifuged at 4°C at 13,000g for 10 min. Supernatant protein concentrations were determined using the Bio-Rad DC Protein Assay Kit (#500-0113, #500-0114, and #500-0115). The 6× SDS sample buffer (#BP-111R, Boston BioProducts) was added to the samples, which were then boiled for 5 min. Ten to forty micrograms of protein was loaded on SDS–polyacrylamide gel electrophoresis gel (#456-1086 and #456-1093, Bio-Rad), transferred to polyvinylidene difluoride membrane (#IPVH00010, Merck Millipore), and probed with primary antibodies overnight. After wash, the membrane was probed with secondary antibodies and developed with enhanced chemiluminescence (#1863096, #1863097, and #34095, Thermo Fisher Scientific).
4
Immunoblotting Analysis of Protein Samples
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Worms were collected in M9 buffer from NGM plates and quickly washed several times. C. elegans or human tissues were suspended in RIPA buffer for sonication and always kept on ice. The lysates after sonication were centrifuged at 10,000g at 4°C to remove the debris and lipids. The supernatants were mixed with equal amounts of protein sample buffer and denatured for 5 min at 95°C. Denatured protein samples were loaded and separated by 4 to 20% SDS–polyacrylamide gel electrophoresis gel (Bio-Rad) and then transferred to polyvinylidene difluoride membranes. The primary antibodies were rabbit anti-HA (hemagglutinin) antibody (Sigma-Aldrich, H6908), anti-TMC1 (Abcam, ab199949), rabbit anti-Phospho-PKC Substrate Motif [(R/K)XpSX(R/K)] MultiMab antibody mix (Cell Signaling Technology, #6967), mouse anti-PKC (Santa Cruz Biotechnology, sc-17769), and rabbit anti-actin (Sigma-Aldrich, A2066) at 1:1000 dilution. The secondary antibody was goat anti-rabbit immunoglobulin G (IgG) at 1:5000 dilution (GE Healthcare). Biotinylated proteins were blotted using horseradish peroxidase–conjugated streptavidin (Thermo Fisher Scientific, N100).
5
Isolation and Analysis of Slick KO Mouse Brain Membranes
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Whole mouse brain membrane isolation by sucrose fractionation was followed according to Bhattacharjee et al.5 (link) Wild-type purebred C57/Bl6 mice were compared with Slick heterozygous (HET) and KO C57Bl6/J/129 mixed strain mice for initial characterization of Slick KO mouse line. Bradford assay was used for loading onto SDS-polyacrylamide gel electrophoresis gel (Bio-Rad), 30 μg total protein per lane. After electrophoresis, the gel was then transferred onto a nitrocellulose membrane and incubated in 5% nonfat dry milk as a blocking agent for 1 hour. Antibody Slick anti-mouse (1:1000; NeuroMab) was used for Western analysis. The signal was detected using Luminata Forte HRP Substrate (Millipore). Densitometric analyses of Western blots were performed by Bio-Rad GS-700 flatbed scanning densitometry. Experiment was repeated twice.
6
Western Blot Analysis of Transcription Factors
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Protein lysates were prepared by washing cells with 1 ml of cold PBS and resuspended in lysis buffer [150 mM NaCl, 50 mM tris-HCl (pH 8.0), 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, and protease inhibitors] for 30 min at 4°C. Lysates were centrifuged at 5°C for 15 min at 13,000 rpm to remove insoluble debris. Protein concentrations were quantified using Pierce BCA (Thermo Fisher Scientific). Proteins were separated by electrophoresis on an SDS–polyacrylamide gel electrophoresis gel (Bio-Rad), transferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific), and blocked with 5% milk in tris-buffered saline (TBS) with Tween 20. The membranes were immunoblotted with anti–c-Myc (Santa Cruz Biotechnology, sc-40), anti–L-Myc (University of Iowa, PCRP-MYCL1-1A3), anti-vinculin (Sigma-Aldrich), anti-ASCL1 (BD Biosciences, #556604), anti-NeuroD1(Proteintech, 12081-1-AP), anti-Smad2 (Cell Signaling Technology, #5339), anti-Rest (Millipore Sigma, 07-579), anti-Hes1 (Santa Cruz Biotechnology, sc-166410), anti-Brn2 (Cell Signaling Technology, 12137), anti-YAP1 (Proteintech, 13584-1-AP). Immunoblots were quantified using ImageJ.
7
Western Blot Analysis of Liver Proteins
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Liver tissue (~50 mg) was homogenized with 500 μl of radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich), 1× protease, and phosphatase inhibitors (Roche). Protein concentration was estimated by bicinchoninic acid (BCA) using bovine serum albumin standard. Protein lysates (80 to 100 μg) were separated on 10% SDS–polyacrylamide gel electrophoresis gel (Bio-Rad) and transferred using a Trans-Blot turbo system (Bio-Rad). After blocking with 5% fat-free milk for 1 hour, membranes were incubated with primary antibodies overnight at 4°C on a shaker, followed by a secondary antibody for 1 hour at room temperature. For glyceraldehyde-3-phosphate dehydrogenase (GAPDH), membranes were directly incubated with GAPDH–horseradish peroxidase antibody for 1 hour and visualized using Amersham Imager 600 (GE Healthcare). Antibodies’ source and dilutions are given in table S4.
8
Western Blot Analysis of ChRas
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Total proteins were isolated from mycelia cultured in PDB for 3 days using a previously described method (Tang et al., 2020 (link)). For Western blotting, proteins were separated on a 12% SDS–polyacrylamide gel electrophoresis gel (Bio-Rad) and electrophoretically transferred to the polyvinylidene fluoride membrane (Millipore, Etobicoke, ON, Canada) in the Bio-Rad Mini Trans-Blot system. ChRas was detected with Anti-KRas Rabbit Monoclonal Antibody (Bioss, Beijing, China). Signals were captured by ChemiDoc XRS + system (Bio-Rad).
9
ZIKV Protein Detection in Infected Cells
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Confluent C6/36 cells grown in a six-well plate were infected with viruses at an MOI of 0.1 or mock infected as control. At 72 h post infection, cell lysates were mixed with loading buffer (0.1 M Tris-HCl (pH 8.8), 20% glycerol, 1% dithiothreitol, and 3% SDS and 0.0025% bromophenol blue), heated for 5 min at 100 °C, cooled on ice, and run on a precast 12% SDS-polyacrylamide gel electrophoresisgel (Biorad, USA). Protein was transferred to polyvinylidene difluoride membranes and the membranes were blocked overnight at 4 °C in PBST (Dulbecco’s PBS + 0.2% V/V Tween 20 + 1% bovine serum albumin). Following overnight blocking, the membranes were incubated for 1 h at room temperature with PBST containing a mouse anti-ZIKV E antibody (1 : 1,000, Cat#BF-1176-56, BioFront Technologies). Membranes were then washed three times with PBST and incubated for 1 h at RT with PBST containing a 1 : 1,000 dilution of sheep anti-mouse horseradish peroxidase (HRP). Membranes were then washed three times with PBST and developed using a Pierce ECL western blotting substrate (Cat#32109, Thermo Fisher Scientific) according to the manufacturer’s recommendation.
10
Western Blot Analysis of Adenosine Receptors
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Equal amounts of protein from cell lysates were mixed with sodium dodecyl sulfate (SDS) sample buffer and separated on SDS–polyacrylamide gel electrophoresis gels (BioRad, Hercules, CA) before Western blot analysis. The primary antibodies used were A2BR (Sigma-Aldrich), A2AR (Sigma-Aldrich), phospho-S6Ser235/236, GAPDH and β-actin (Cell Signaling Technology, Danvers, MA). Quantification of the Western blot data were performed by measuring the intensity of the hybridization signals by using ImageJ analysis software (National Institutes of Health, Bethesda, MD). Cells were cultured in 10 mL of RPMI-1640 medium with 10% FBS in the presence or absence of NECA, ZM 241385 or PSB 603. Cells were lysed in RIPA buffer supplemented with Complete Mini Ethyl-enediaminetetraacetic Acid–Free Protease Inhibitor Cocktail (Roche) and PhosSTOP (Roche), and their protein concentrations were determined by using a BCA protein assay kit (Pierce; ThermoFisher Scientific).
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