To prepare the solution for SDS-PAGE, 1 × 1 cm2 of the protein adsorbed fabric was placed into a 1.5 mL microtube that contained 0.5 mL of 1x PBS. Then, the microtube containing protein-adsorbed fabric was shaken using a micro-shaker at 500 rpm. The solution was kept every 1 h to observe the detachment of the entrapped protein on the fiber surface. SDS-PAGE was used to investigate whether the sample retained the S-protein residues. A running buffer solution for SDS-PAGE was prepared by mixing 100 mL of 10x Tris/Glycine/SDS buffer (Bio-Rad, Hercules, CA) with 900 mL distilled water. S-protein, a positive control, and washing solution from each fabric samples 12 μL were mixed with 4x SDS sample buffer (Merck, Darmstadt, Germany) 3 μL. The mixtures were heated at 95 °C for 5 min. All the samples were loaded on an Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-Rad). Precision Plus Protein™ Dual Color Standards (Bio-Rad) was used as a protein ladder standard. Electrophoresis was performed for 60 min using the Tris/Glycine/SDS buffer at a constant voltage of 100 V. Imaging was performed after transferring the gel to Gel Doc™ EZ Imager (Bio-Rad). Image analysis of the stain-free gel was performed using the Image Lab software (Version 6.1.0, Bio-Rad).
Tris glycine sds buffer
Manufactured by Bio-Rad
Sourced in United States, Japan, France, United Kingdom
Tris/Glycine/SDS buffer is a pre-made solution used in gel electrophoresis applications. It provides the necessary ions and pH for the separation and migration of proteins or nucleic acids in a polyacrylamide or agarose gel matrix.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli sample buffer, by Bio-Rad (26 mentions)
Tris glycine buffer, by Bio-Rad (13 mentions)
Mini protean tgx gel, by Bio-Rad (10 mentions)
Clarity western ecl substrate, by Bio-Rad (10 mentions)
Mini protean tgx precast gel, by Bio-Rad (9 mentions)
Image lab software, by Bio-Rad (7 mentions)
Laemmli buffer, by Bio-Rad (7 mentions)
Trans blot turbo transfer system, by Bio-Rad (6 mentions)
Blotting grade blocker, by Bio-Rad (5 mentions)
Precision plus protein dual color standard, by Bio-Rad (4 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (4 mentions)
Mini protean tetra cell, by Bio-Rad (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Mini protean tgx, by Bio-Rad (4 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Trans blot turbo system, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Mp tgx stain free gel, by Bio-Rad (3 mentions)
103 protocols using tris glycine sds buffer
1
Protein Detachment and SDS-PAGE Analysis
2
SDS-PAGE Analysis of Protein Extracts
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For SDS–PAGE analysis, 100 µL of protein extract were precipitated by adding 400 µL of acetone for 20 min at cold conditions. The proteins were collected by centrifugation at 10,000× g for 10 min at 4 °C, and the resulting pellet was dissolved in 50 µL of Laemmli sample buffer containing 5% (v/v) 2-mercaptoethanol, according to [23 (link)]. In order to determine the molecular weight of the extracted protein products, their separation was carried out on Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad). Electrophoresis was performed at constant voltage (200 V) using Tris/Glycine/SDS buffer (Bio-Rad) as running buffer. Then, gels were stained during 1.5 h with Coomassie Brilliant Blue R-250 staining solution (Bio-Rad). Finally, gels were washed with a solution composed of water/methanol/acetic acid (60%:40%:10%, v/v) overnight. The molecular mass markers Precision Plus Protein™ Standard Unstained (10–250 kDa) (Bio-Rad) were used.
3
Protein Extraction and Western Blot
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Cells were harvested 1 and 5 h after treatment. Cells were washed with PBS, harvested into SDS sample lysis buffer (SLB; 2% SDS, 63 mM Tris–HCl, pH 6.8, and 10% glycerol), sonicated, and centrifuged. Protein concentration was determined by BCA assay (Pierce Biotechnology, Rockford, IL, USA); samples were adjusted to equal protein amount (40 μg) with SLB containing dithiothreitol (1% final conc.) and bromophenol blue (0.02% final conc.), and separated by SDS–PAGE in Tris-glycine-SDS buffer (BioRad, 1610772). Proteins were electrotransferred onto a nitrocellulose membrane (0.45 μm NC, Amersham™, GE Healthcare Life Sciences) using wet transfer in Tris-glycine buffer (BioRad, 1810704) with 10% methanol (Sigma, 59304). After blocking with 5% non-fat milk in PBS/Tween-20, proteins were detected using specific antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. Peroxidase activity was detected by ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA).
4
Western Blot Analysis of Shh-Signaling Proteins
5
Western Blot Analysis of Protein Samples
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Cells were washed with PBS, harvested into SDS sample lysis buffer (SLB; 2% SDS, 63 mM Tris-HCl, pH 6.8, 10% glycerol), sonicated, and centrifuged. Protein concentration was determined by BCA (Pierce Biotechnology, IL, Rockford, USA), samples adjusted to equal protein amount (40 μg) with SLB containing DTT (1% final conc.) and bromophenol blue (0.02% final conc.) and separated by SDS–PAGE in Tris-glycine-SDS buffer (BioRad, 1610772). Proteins were electrotransferred onto a nitrocellulose membrane (0.45 μm NC, Amersham™, GE Healthcare Life Sciences) using wet transfer in Tris-glycine buffer (BioRad, 1810704) with 10% methanol (Sigma, 59304) and after blocking with 5% non-fat milk in PBS/Tween-20 were detected using specific antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. Peroxidase activity was detected by ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative analysis was done by Image J 1.48v program with GAPDH as the internal control.
6
Protein Extraction and Electrophoresis
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Cells were washed once with phosphate‐buffered saline (PBS), scraped, lysed in RIPA lysis buffer (MilliporeSigma, Billerica, MA, USA; Cat #20‐188) containing a protease inhibitor cocktail (Roche, Cat #11697498001) and lysates incubated on ice for 30 minutes. The protein concentration of samples was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Cat #23227). Cell lysates were prepared for electrophoresis by addition of SDS sample buffer (Invitrogen, Carlsbad, CA, USA; Cat #NP0007) containing 2.5% β‐mercaptoethanol and heated to 95°C for 10 minutes. Forty micrograms of protein were loaded onto 4%‐20% gradient Mini‐PROTEAN TGX gels (BioRad Laboratories, Hercules, CA, USA; Cat #456‐1093). A protein marker (Gel Company, San Francisco, CA, USA; Cat #FPL‐008) was used as a standard. Proteins were separated by electrophoresis using Tris/Glycine/SDS buffer (BioRad Laboratories, Cat #161‐0732).
7
Protein Expression Analysis of Cell Lines
8
Adult Oyster Histamine Receptor Analysis
9
Western Blot Protein Extraction and Analysis
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Cells were washed with DPBS and lysed with Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA; catalog #: 78501) with 1X Halt's Protease and Phosphatase (Thermo Fisher Scientific, Waltham, MA, USA; catalog #: 78440) by incubation for 3 min. Cell lysate was collected and stored at −80 °C until used. Samples were mixed with Laemmli sample buffer (BioRad, Hercules, CA, USA; catalog #: 1610737) at a working concentration of 1X and incubated at 97 °C for 5 min. Samples were loaded into a pre-cast MP TGX stain free gel (BioRad, Hercules, CA, USA; catalog #: 4568095) and run at 200V for 30 min in 1X Tris/Glycine/SDS buffer (BioRad). Protein was transferred to a PVDF membrane using a Trans-blot Turbo Transfer System (BioRad). The membrane was blocked for 30 min at room temperature in 1X TBST+5% Dry Milk. The membrane was incubated overnight at 4 °C with primary antibodies and for 1 h at room temperature with secondary antibodies in 1X TBST+5% Dry Milk. The membrane was washed between each antibody exposure with 1X TBST. Chemiluminescence was activated using Clarity Western ECL Substrate (BioRad) and the blot was imaged using a ChemiDoc Touch Imaging System and Image Lab software (BioRad).
10
Western Blot Analysis of Protein Extracts
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Protein extracts in RIPA were separated by SDS-PAGE using pre-cast gels (4–20%, Bio-Rad) in Tris–Glycine-SDS buffer (Bio-Rad, Spain). Protein was transferred to nitrocellulose membranes (Bio-Rad) using the TransBlot Turbo system from Bio-Rad. Membranes were subsequently blocked for 1 h with skim milk at 5% (w/v) in PBS and then washed three times for 10 min each in PBS supplemented with 0.1% (v/v) Tween 20 (PBS-T). Membranes were then incubated with primary antibodies in PBS-T overnight. Following this, the membranes were incubated with secondary antibodies for 2 h. After the secondary antibody incubation, membranes were washed three times with PBS-T. According to the manufacturer’s protocol, the membranes were developed using SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoScientific, Spain) and imaged using a ImageQuant LAS-4000 biomolecular imager (GE Healthcare).
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