Reagents included: MTT, Tiron, aminoguanidine bicarbonate (AG) and LPS (Sigma-Aldrich (Milan, Italy); Laemmli buffer (Thermo Fisher Scientific, Monza, Italy); BCA kit (Pierce, WA, USA).
Laemmli buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States, Italy, United Kingdom
Laemmli buffer is a commonly used buffer solution in biochemistry and molecular biology laboratories. Its core function is to prepare protein samples for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The buffer contains SDS, which denatures proteins and gives them a uniform negative charge, and reducing agents that break disulfide bonds, allowing for the separation of proteins by their molecular weight.
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Lab products found in correlation
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Supersignal west dura substrate, by Thermo Fisher Scientific (2 mentions)
Anti ptgs2, by Abcam (2 mentions)
Imagej, by Bio-Rad (2 mentions)
Image lab, by Bio-Rad (2 mentions)
Anti vegf, by Abcam (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Irdye 680cw goat anti mouse, by LI COR (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye 800cw goat anti rabbit, by LI COR (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Roti block, by Carl Roth (2 mentions)
Nupage gel, by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Mes buffer, by Thermo Fisher Scientific (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
39 protocols using laemmli buffer
1
Oxidative Stress Assay Protocol
2
Western Blot Protein Extraction
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Extraction of proteins was realized in RIPA buffer (#89900; Thermo Fisher Scientific) associated with phosphatase and protease inhibitors (#88669; Thermo Fisher Scientific). Protein content was measured by Bio-rad laboratory DCTM Protein Assay reagent A, B and S (#500-0113, #500-0114 and #500-0115). A minimum of 15 µg of protein was solved in Laemmli buffer (Thermo Fisher Scientific), denaturated at 100°C and separated by electrophoresis using 4%–12% Bis-Tris gels (Thermo Fisher Scientific) in MES buffer (Thermo Fisher Scientific). Then, proteins were transferred to nitrocellulose membrane (Merck Millipore IPVH00010) in transfer buffer (25 mM Tris; 190 mM glycine; 20% methanol in H2O) at 200 mA for 1.5 hour. Membranes were washed in Tris-buffered saline with Tween20 buffer (TBST; 20 mM Tris, pH 7.5 150 mM NaCl 0.1% Tween 20 in H2O) and treated with blocking buffer (5% bovine serum albumin (BSA) in TBST) for 1 hour. Membranes were exposed to primary antibody diluted in 5% BSA in TBST overnight at 4°C. The day after membranes were washed with TBST and incubated with appropriate horseradish peroxidase-coupled secondary antibody (Southern Biotech, Birmingham, Alabama, USA) for 1 hour at room temperature. Then proteins were revealed with ECL (GE Healthcare, Chicago, Illinois, USA). β-actin (1:10,000) was used to verify equal protein loading.
3
Analytical Reagents in Cell Assays
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All of the chemicals used in the present study were analytical grade reagents from various sources. Phosphate-buffered saline (PBS), formaldehyde acetic acid and ammonium hydroxide, aminoguanidine bicarbonate (AG), MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and FPS-ZM1 were purchased from Merck Spa (Milan, Italy). Laemmli buffer and 2′–7′-dichlorofluorescein-diacetate (H2DCF-DA) were purchased by Thermo Fisher Scientific (Milan, Italy), Roti-Block from Prodotti Gianni (Milan, Italy), and bicinchoninic acid (BCA) kit from Thermo Fisher Scientific (Milan, Italy).
4
Quantifying IL-6Rα Expression in Tissue
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MGC lysates and tissue homogenates were prepared in radioimmunoprecipitation (RIPA) buffer (Cell Biolabs, Inc., San Diego, CA, USA) containing protease and phosphatase inhibitors (Sigma-Aldrich). Equal amounts of protein (20–40 µg per sample) were denatured in Laemmli buffer (Thermo Scientific), heated to 100 °C for 10 min, and then loaded in a pre-cast 4–15% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) (Bio-Rad) for electrophoresis. Proteins were separated at 100–120 V and transferred to a nitrocellulose membrane, and the membrane was blocked in 5% milk in TBST for 2–3 h at room temperature. After blocking, the membrane was incubated with anti-IL6Rα antibody (MA5-29721, Invitrogen, 1:500) at 4 °C overnight. The membrane was washed 3 times in TBST and then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (31460, Invitrogen) for ~ 2 h at room temperature. The membrane was washed and then developed with ECL substrate (Thermo Scientific), and images were captured with the ChemiDoc Imaging System (Bio-Rad). Bands were quantified using ImageJ software (NIH), and IL-6Rα expression values were normalized to the expression of β-actin (AC028, ABclonal). Quantified western blot data was presented as fold control relative to normalized wildtype expression.
5
Immunoprecipitation Assay of OAT3 Protein
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The immunoprecipitation assay of OAT3 was conducted based on the standard protocol established in our lab [13 (link),14 (link)]. In short, treated cells were lysed at 4 °C, and an equal amount of total proteins (~1000 μg) was mixed with protein G resin at 4 °C for 4 h to lower non-specific binding. In the meantime, the primary antibody was mixed with protein G resin at 4 °C to create an antibody–resin complex. Then, the pre-cleared protein samples were incubated with the antibody–resin complex at 4 °C overnight with gentle shaking. Next, the proteins captured on protein G resin were denatured and eluted with Laemmli buffer from Thermofisher Scientific (Waltham, MA, USA). The eluted samples were measured by standard SDS-PAGE and western blotting with indicated primary and secondary antibodies in each experiment.
6
SDS-PAGE Analysis of Elastin-Like Polypeptides
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The purity of ELPs was evaluated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). A total of 10 μg per 15 μL of ELPs were mixed with β-mercaptoethanol in Laemmli buffer (Thermo Fisher Scientific, Waltham, MA, USA #31350010 and Bio-Rad, Hercules, CA, USA #1610747, respectively) at a 1:4 ratio, denatured at 95 °C for 5 min, loaded on a 4−20% gradient Mini-Protean TGX precast gel (Bio-Rad, Hercules, CA, USA #456-1095), and run for 32 min at 200 volts. The gel was stained by G-250 Coomassie (Bio-Rad, Hercules, CA, USA #1610786) and imaged by ChemiDoc Touch Image System (Bio-Rad laboratories, CA, USA).
7
EGFR Protein Immunoprecipitation Protocol
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Cells were lyzed in RIPA buffer (Beyotime Institute of Biotechnology), and 500 μg of protein was incubated with an anti-EGFR antibody for 1 h at 4°C. Antibodies were precipitated using Protein G-coated Dynabeads (Thermo Fisher Scientific), and bound protein was eluted in Laemmli buffer (Thermo Fisher Scientific) for 10 min at 70°C.
8
Choroidal Protein Expression Analysis
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Choroidal lysates were prepared from samples of the C57BL/6 mice taken at the indicated time interval after laser injury. Protein samples, quantified using NanoDrop 2000 UV-Vis Spectrophotometer (30 µg/each lane; Thermo Fisher Scientific, Inc.) were dissolved in Laemmli buffer (cat. no. 1610737; Bio-Rad Laboratories, Inc., Hercules, CA, USA), boiled for 3–4 min, and centrifuged at 4°C for 2 min at 20,000 × g to remove insoluble materials. A total of 30 µg protein per lane was separated by SDS-PAGE (12%) and transferred to 0.2 µm nitrocellulose membranes. The blocked membranes were probed overnight (4°C) with goat anti-VEGF (1:200; sc-152-G; Santa Cruz Biotechnology, Inc.), goat anti-HIF-1α (1:200; sc-12542Y-15; Santa Cruz Biotechnology, Inc.), goat anti-SDF-1α (1:200; sc-7427; Santa Cruz Biotechnology, Inc.), goat anti-CCL3 (1:1,000; AB-450-NA; R&D Systems) or goat anti-GAPDH antibodies (1:200; sc-48166; Santa Cruz Biotechnology). The membranes were then incubated at RT for 1 h with a horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (1:5,000; 5160–2504; R&D Systems), and immunoreactive bands were visualized using ECL reagent (Advansta, Menlo Park, CA, USA). The intensities of the protein bands were quantified and normalized to GAPDH using Image J Software version 2.1.4.7 (National Institutes of Health, Bethesda, MD, USA).
9
Immunoprecipitation and Western Blotting
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Protein lysates were prepared in lysis buffer, on ice and incubated for 30 min, followed by centrifugation at 15,000 rpm for 15 min at 4 °C. For immunoprecipitation (IP). the precipitated proteins were collected using A+G beads, followed by washing and eluting using Laemmli buffer (Thermo Fisher Scientific, Waltham, MA, USA), and finally subjected to western blotting. Same amount of protein was loaded on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). The membranes were probed overnight at 4 °C with primary antibodies purchased from Abnova (Beijing, China) and Cell Signaling Technology. Blots were then incubated with HRP conjugated secondary antibodies and developed with ECL reagents (Invitrogen, USA).
10
Isolation of 3xHA-E2F6 Complexes
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ES cell line E14TG2a was transfected with the pCAG-3×HA-E2F6 vector and selected with blasticidine at 0.01 µg/µL until stable integration of the plasmid. Nuclear extract (1 mg) was incubated with anti-HA magnetic beads (Pierce) overnight at 4 °C. The complex bead–proteins was washed in a buffer containing 15 mM of Tris pH 8, 150 mM NaCl, 0.05% Triton. Proteins were eluted in Laemmli buffer (Thermo Fisher Scientific). As a control, immunoprecipitation was performed on E14TG2a cells not expressing 3×HA-E2F6. Twenty-five percent of eluted proteins and 15 µg of input proteins were loaded on a 10% SDS-polyacrylamide gel and assessed by western blotting.
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