Nuclear and cytoplasmic protein lysates were isolated from MDA-MB-231 bone cells after 72 hr treatment with GANT58 (0–20 μM) using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Protein concentrations were quantified using the Pierce BCA Protein Assay Kit (Thermo Scientific). Protein samples (20 μg/well) were separated on a 4–20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad) by SDS-PAGE prior to being transferred to a nitrocellulose membrane (Bio-Rad) with the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were then blocked for 1 hr in 1X TBS containing 0.1% Tween-20 and 5% w/v BSA and incubated with the following primary antibodies at 4°C overnight: anti-Gli2 (1:500, Novus Biologicals), anti-TATA binding protein (TBP) (1:1000, Cell Signaling), or anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000, Cell Signaling). Following incubation with anti-rabbit IgG HRP-linked secondary antibody (Cell Signaling) at room temperature for 1 hr, protein bands were developed by Western Lightning Plus-ECL (Perkin Elmer) and imaged on a ChemiDoc MP Imaging System (Bio-Rad). The intensity of each band was determined by densitometry using ImageJ software.
Mini protean tgx polyacrylamide gel
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN TGX polyacrylamide gels are a precast gel system designed for protein electrophoresis. They are manufactured using a proprietary formulation to provide consistent and reliable separation of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Ripa buffer, by Merck Group (4 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (3 mentions)
Trans blot turbo pvdf membrane, by Bio-Rad (3 mentions)
Laemmli buffer, by Bio-Rad (3 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Blotting grade blocker, by Bio-Rad (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Anti tata binding protein tbp, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Precision plus protein westernc standard, by Bio-Rad (1 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (1 mentions)
24 protocols using mini protean tgx polyacrylamide gel
1
Quantifying Nuclear and Cytoplasmic Proteins in MDA-MB-231 Cells
2
Western Blot Analysis of ER Stress Markers
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For Western blot analysis, a 4–20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad Laboratories) was used. Each sample was then denatured at 94°C for 3 min before being loaded. Gels were blotted onto TransBlot Turbo polyvinylidene fluoride membranes (Bio-Rad Laboratories), and blocked in PBST buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.1% Tween 20, pH 7.42) containing 5% nonfat dry milk. The signal was measured by chemoluminescence (ECL plus; GE Healthcare). Western blots were performed using rabbit anti-mouse HERP (Santa Cruz Biotechnology, Inc.), CHOP, BIP, IRE1α (Cell Signaling Technology), goat anti–mouse Igκ (Beckman Coulter), and using goat anti–mouse GAPDH (R&D Systems) Abs for normalization.
3
SDS-PAGE Analysis of Phage Proteins
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Two-fold serial dilutions of isolated phage protein in 1X Laemmli sample buffer were heated at 95°C for 1 h. A 10-fold dilution of the Precision Plus Protein WesternC Standards (Bio-Rad, Hercules, CA) was diluted in 1X Laemmli sample buffer and heated at 95°C for 10 min. Prepared samples were separated on a 4–20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad, Hercules, CA) at a constant voltage of 100 V for 45 min. Following separation, the gel was fixed for 10 min in a methanol/acetic acid/water (v/v/v 50:10:40) fixing solution. Protein bands were labeled with a Colloidal Blue Staining Kit (Thermo Fisher Scientific, Waltham, MA) as described in the manufacturer's instructions. Briefly, the fixed gel was incubated in the prepared staining solution (stainer A/stainer B/methanol/water v/v/v/v 20:5:20:55) for 3 h at room temperature with gentle shaking. The staining solution was replaced with distilled water and destained for 11 h at room temperature with gentle shaking. Following destaining, bands were visualized using an EDAS Imaging Station (Eastman Kodak Co., Rochester, NY) equipped with a DC290 digital camera and a white light box. Images were captured and analyzed using Molecular Imaging Software (v. 4.0.3; Eastman Kodak Co., Rochester, NY).
4
Western Blot Analysis of SCN9A Protein
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A431 or HEK293 cells were lysed in RIPA buffer (Sigma-Aldrich, #R0278) with protease inhibitors (Sigma-Aldrich, #P8340). Protein concentrations were measured using the Coomassie Plus Bradford assay kit (Thermo Scientific, #23236). For Western blot, 15 µg of whole cell lysates were mixed with loading buffer and boiled for 5 min, and then were run in 4–15% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad, #456-1083). After transferring, the nitrocellulose membranes were first blotted with 50 mM Tris, 150 mM NaCl and 0.1% Tween-20 (TBS-T) with 5% nonfat milk for 30 min at room temperature. The membranes were then incubated with either anti-SCN9A monoclonal antibody (1:500 dilution in TBS-T, Millipore, #MABN41) or anti-actin monoclonal antibody (1:5,000 dilution in TBS-T, Millipore, #MAB1501) at 4°C overnight. The membranes were then washed with TBS-T three times, each time for 10 min. Subsequently, the membranes were incubated with goat anti-mouse IgG (H+L) secondary antibody (1:10,000 dilution in TBS-T, Thermo Scientific, #31430) at room temperature for 45 min. The membranes were then washed with TBS-T four times, each time for 10 min. The membranes subsequently were incubated with ECL Western blotting substrates (Thermo Scientific, #32106) and then exposed to X-ray films (Sigma-Aldrich, #Z370398).
5
Protein Separation and Visualization
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A total of 40 µg of the pre-treated and post-treated CSMPI was added to 2× Laemmli buffer (Bio-Rad, Hercules, CA, USA) with 50 mM dithiothreitol (DTT) and heated at 95 °C for 5 min; then, they were loaded into a 12% pre-cast Mini-PROTEAN® TGX™ polyacrylamide gel (Bio-Rad, CA, USA) for protein separation. The electrophoresis was set at 200 V, and the gel was run for 35 min. Protein bands were visualised using Coomassie Blue stain, and the gel image was captured using the GE Image Scanner III (Ge Healthcare, Chicago, IL, USA).
6
Protein Extraction and Western Blot Analysis
7
Western Blot Analysis of Phospho-AMPK in Mouse Liver
8
Protein Extraction and Western Blot Analysis
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Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Sigma #R0278) supplemented with halt protease and phosphatase inhibitor cocktail (Thermo Scientific # 78442). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific #23223) per the manufacturer’s instructions. Equal amounts of protein extracts were separated using 4–15% mini-Protean TGX polyacrylamide gel (Bio-Rad #456–1033). Samples were transferred to a Trans-Blot Turbo PVDF membrane (Bio-Rad #1704156). The membrane was blocked using Blotting-Grade Blocker (Bio-Rad #170–6404) dissolved in TBST and probed with varying primary antibodies. The membrane was then treated with HRP conjugated secondary antibodies. The immunoreactivity was detected with either the Pierce ECL Western Blotting Substrate or the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Densitometric analysis was performed with ImageJ, and relative values were displayed under respective blots.
9
Myosin Expression Quantification in Drosophila
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Relative levels of myosin expression were determined by measuring myosin-to-actin ratios in upper thorax homogenates subjected to one-dimensional SDS polyacrylamide gel electrophoresis (O'Donnell et al., 1989 (link)). Six dissected upper thoraces from 2-day-old adult female flies for each line were homogenized in 360 μl of SDS gel 1× Laemmli Sample Buffer (Bio-Rad, Hercules, CA) containing 5% 2-mercaptoethanol. Samples were boiled for 5 min prior to loading at 2-5 μl on a 15-well 10% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad). Gels stained with Coomassie Brilliant Blue R-250 were digitally scanned using an Epson Expression 636 flatbed scanner. Myosin heavy chain and actin levels were determined by UN-SCAN-IT gel software (Silk Scientific) for three separate lanes per sample. The median myosin-to-actin ratio in mutant thoraces was compared with the median ratio found in thoraces from aged-matched flies expressing the PwMhc2 control transgene in the corresponding genetic background.
10
Analysis of Quorum Sensing Regulators in Pseudomonas aeruginosa
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The PA14 csy4-3×flag and ΔlasI ΔrhlI csy4-3×flag mutant strains were streaked onto LB medium and grown at 37, 30, 23, or 15°C until individual colonies reached 1 mm in diameter. Single colonies were harvested and lysed with BugBuster protein extraction reagent (Millipore), following the manufacturer’s instructions. Fifty micrograms of protein was separated by SDS-PAGE on a 4 to 20% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad) and blotted onto a polyvinylidene difluoride (PVDF) membrane (catalog no. 1620174; Bio-Rad). The membrane was incubated for 1 h with monoclonal Anti-FLAG M2-peroxidase (HRP) antibody (catalog no. A8592; Sigma), monoclonal anti-RpoB antibody (catalog no. ab191598; Abcam), both at a 1:3,000 dilution, or polyclonal anti-GroEL antibody (catalog no. G6532; Sigma) at a 1:15,000 dilution in Tris-buffered saline with Tween 20 (TBST) and 5% skim milk. Anti-rabbit antibody (catalog no. W4011; Promega) was used as the secondary antibody for detection of the anti-RpoB and anti-GroEL antibodies. The membrane was washed in TBST and was developed using SuperSignal West Femto maximum sensitivity substrate (catalog no. 34095; Thermo Scientific).
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