DMEM, Hank's-balanced salt solution (HBSS), D-PBS, trypsin-ethylenediaminetetraacetic acid solution, metformin, compound C, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT), 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), SB202190, SP600125, wortmannin, rapamycin, H33342, and acridine orange (AO) were purchased from Sigma Chemical Corp. (St. Louis, MO, USA). FBS was obtained from Life Technologies, Inc. (Rockville, MD, USA). Polyclonal antibodies against poly ADP ribose polymerase (PARP), cleaved caspase-3, autophagy-related (3, 5, 7), β-actin, and monoclonal antibodies against beclin-1 and microtubule-associated protein 1 light chain 3B (LC3B) were purchased from Cell Signaling Technology (Danvers, MA, USA). Polyclonal antibodies against phospho-AMPK were obtained from Millipore (Billerica, MA, USA). Polyclonal anti-PARP and anti-rabbit goat immunoglobulin G-horseradish peroxidase (HRP) secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Electrophoresis reagents (including Bis-Tris gels, running buffer, and polyvinylidene difluoride [PVDF] membranes) were obtained from Invitrogen (Carlsbad, CA, USA).
Running buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
Running Buffer is a laboratory solution used to provide the appropriate pH and ionic conditions for the electrophoretic separation of biomolecules, such as proteins or nucleic acids, in gel-based analysis techniques. It serves as the medium for the movement and separation of these molecules during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (3 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Transfer buffer, by Bio-Rad (2 mentions)
Tbs t buffer, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
Xbp1s, by Cell Signaling Technology (1 mentions)
Catalase, by Nanjing Jiancheng (1 mentions)
Total superoxide dismutase t sod, by Nanjing Jiancheng (1 mentions)
Polyvinylidene fluoride pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Phosphor perk, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Malondialdehyde, by Nanjing Jiancheng (1 mentions)
Glutathione peroxidase gsh px, by Nanjing Jiancheng (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
9 protocols using running buffer
1
Investigating Cellular Stress Pathways
2
Investigating Metformin's Effects on ER Stress Signaling
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All the solvents used for extract were of analytical grade (Baishi Chemical Co. Ltd., Tianjin, P. R. China), and water was double distilled. Metformin hydrochloride tablets were purchased from Harbin Tongyitang Pharmaceutical Co., Ltd (China). Antibodies to GRP78, ß-actin, phospho-IER1α, phosphor-PERK, phospho-e1F2α, XBP1s, IER1α, PERK, and e1F2α were obtained from Cell Signaling Technology (Danvers, MA, USA). The secondary antibody was purchased from Boster Biological Technology Co., Ltd (China). Electrophoresis reagents, including Bis-Tris gels, running buffer, and poly (vinylidene fluoride) (PVDF) membrane, were obtained from Invitrogen (Carlsbad, CA, USA). ECL was bought from GE Healthcare (UK). Kit of low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), blood plasma free fatty acid (FFA), total cholesterol (TC), triacylglyceride (TG), fructosamine, hexokinase (HK), glycogen, catalase (CAT), total Superoxide Dismutase (T-SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (China). Pierce TMT® Mass Tagging Kits and Reagents were purchased from Thermo Science Pierce (Rockford, USA).
3
Western Blot Analysis Methodology
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Western blot was performed as we previously described.70 (link) Cells were lysed in sample buffer [63 mM tris (pH 7.0), 10% glycerol, and 2% SDS in nuclease free water]. Cell lysates were heated at 95°C for 10 min and sonicated briefly. Cell debris were removed by centrifugation at 12000 rpm for 5 min at 4°C. The supernatant was subjected to 8% SDS-PAGE in running buffer (Invitrogen Cat.B0002–02), followed by Western blot. Proteins were transferred to PVDF membrane with 0.45 μm pore size (Millipore, Cat. IPVH00010) in transfer buffer (Bio-Rad, Cat. 161–0771). The membrane was blocked with 5% nonfat milk in TBS-T buffer (Thermo Fisher, Cat. 28360) supplemented with 1% bovine serum albumin (HyClone, Cat. SH30574.02) at room temperature for 1 h. Primary and secondary antibodies were diluted with TBS-T supplemented with 1% BSA. Incubation of antibodies was performed for 1 h at room temperature. Membrane was washed 5 times for 5 min each time with TBS-T. LI-COR Odyssey imaging system was used to detect the chemiluminescence signal. Quantification of protein band intensity was determined by generating a ratio of mean intensity over median intensity using Adobe Photoshop (CS4). Results were normalized to β-actin.
4
Western Blot Analysis Methodology
5
Western Blot Protein Quantification
6
Western Blot Analysis of Protein Expression
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NTera2 cells were resuspended in 50 μl of radioimmunoprecipitation assay (RIPA) buffer with protease inhibitors (Roche, Baser, Switzerland). Bradford assay was used to quantify the amount of protein in each sample and Western blot was performed with 20 μg of protein in each lane. Protein was loaded into wells of a NOVEX SDS/PAGE (Life Technologies) gel, which was run at 150 mA with running buffer (Thermofisher scientific). Protein was transferred to an Amersham Hybond ECL nitrocellulose membrane (GE Healthcare Lifesciences, Buckinghamshire, UK) at 400 V, 250 mA and 50 W for 90 min. Membranes were then blocked with 5% skimmed milk powder in PBST (PBS + Tween®20) for 30 min at RT, followed by incubation overnight with the relevant primary antibody diluted in 5% skimmed milk in PBST at 4 °C. The following day membranes were incubated for 30 min at RT with a secondary antibody conjugated with IRDye 680 or 800 (LI-COR Biosciences, Nebraska, USA) at a concentration of 1 in 10,000, and scanned in the LI-COR Odyssey scanner (LI-COR Biosciences). Images were captured by Image Studio™ (Li-COR Biosciences) software. Tubulin or Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detection were used as loading controls. Relative protein expression was quantified with Image Studio™.. Primary antibody details can be found on Table 1 .
7
Western Blot Analysis of NLRP3 and IL-1β
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For WB analysis, an equal amount of total protein (20–50 μg) was loaded onto a 12% Tris-Glycine Gel in the NuPAGE 2-morpholino-ethanesulfonic acid (MES) sodium dodecyl sulfate (SDS) Running Buffer (Thermo Fisher Scientific, MA, USA) and then transferred by using the iBlot 2 NC Stack System (Thermo Fisher Scientific, MA, USA). The membranes were blocked in 5% non-fat milk in TBST for 1 h at room temperature and probed with primary antibodies (NLRP3, 1:1,000; Novus Biologicals, 12,446, CO, USA; IL-1β, 1:1,000; R&D Systems, AF-401, MN, USA) overnight at 4°C. Species appropriate secondary antibodies conjugated to IRDye 680RD were used according to the instructions of the manufacturer. Band intensities were quantified by the Image J Software and quantification on each band was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
8
Native Gel Electrophoresis and Protein Transfer
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Proteins extracted as described above were mixed with a loading buffer (20 mM KOAc, 0.006% bromophenol blue, and 10% glycerol) and kept on ice. Proteins were separated on a SuperSep Ace gel using the Novex Tris-Glycine Native Running Buffer (Thermo, LC2672). The gel was incubated in a transfer buffer containing 0.05% SDS and subjected to protein transfer as described in Immunoblotting.
9
Western Blot Protein Detection
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Running Buffer (Thermo Fisher Scientific, Carlsbad, CA, USA) according to the manufacturer's protocol. The samples were then transferred to polyvinylidene difluoride membranes (Trans-Blot Turbo Transfer Pack ® ; Bio-Rad, Hercules, CA, USA). After blocking the membranes with skim milk, they were incubated with primary antibody for RECK (ab88249; Abcam, Cambridge, MA, USA) and β-actin (#4863; Cell Signaling Technology, Danvers, MA, USA) using SNAP i.d. (Merck, Darmstadt, Germany). We used an enhanced chemiluminescence system (ImageQuant LAS 4000 mini; General Electric, Fairfield, CA, USA) to detect the bands.
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