Adherent cells and spheroids were cultured for 48 h under normoxic and hypoxic conditions. Cells were lysed in radioimmunoprecipitation buffer supplemented with protease and phosphatase inhibitors. Protein concentrations in the lysates were quantified using the Pierce Bicinchoninic acid assay (Thermo Fisher Scientific). Equal protein concentrations were loaded in a 4% stacking and 10% SDS separating gel. Proteins were transferred onto a PVDF membrane (Bio-Rad) and blocked with 5% milk in TBST. Primary antibodies were used against DRP1, OPA1 (Novus Bio), MFN1, FIS1 (Protein Tech), UCP2 (Santa Cruz), UCP3 (ThermoFisher Scientific), TOMM 20 (Millipore), and superoxide dismutase 2 (SOD2) antibody (Cell Signaling). Proteins were normalized using the ribosomal protein L19 (L19) or to total protein normalization substrate (Thermo Fisher Scientific). Blots were subsequently probed with the appropriate HRP-conjugated mouse and rabbit or IRDye 680/800cw (Licor) secondary antibodies Proteins were visualized using chemiluminescence Pico ECL (ThermoFisher Scientific) solution with an exposure of 15–30s using the transilluminator from Bio-Rad or the Licor Odyssey Clx imaging system. Proteins quantified using ImageJ software. Data presented as mean ± SEM from at least three biological replicates.
Pierce bicinchoninic acid assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce bicinchoninic acid (BCA) assay is a colorimetric method used to quantify total protein concentration in a sample. It is a two-step process that combines the reduction of Cu2+ to Cu+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid.
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Lab products found in correlation
Protease and phosphatase inhibitor tablet, by Thermo Fisher Scientific (3 mentions)
Total protein normalization substrate, by Thermo Fisher Scientific (2 mentions)
Tomm20, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Bio plex cell lysis kit, by Bio-Rad (2 mentions)
Branson sfx150, by Emerson (2 mentions)
Pro luminex assay, by Thermo Fisher Scientific (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Irdye 680 800cw, by LI COR (1 mentions)
Mops running buffer, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Pentobarbital, by Akorn (1 mentions)
32 protocols using pierce bicinchoninic acid assay
1
Mitochondrial dynamics in hypoxia
2
Multiplex Cytokine Profiling in Murine Ileitis
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Concentrations of plasma cytokines and chemokines (IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES, and TNF-α) were determined in all mice (n = 163, excluding n = 4 used in flow cytometry studies) as previously described (9 (link)). Concentrations of cytokines and chemokines (identical to those measured in plasma, with the addition of IL-33) in 2 cm of ileum tissue were determined in the 153 mice not used for transmission studies (n = 10) or flow cytometry studies (n = 4). Ileum tissue was collected at necropsy, weighed, snap-frozen, and stored at −80° C until processing for protein isolation using the Bio-Plex cell lysis kit (Bio-Rad, Hercules, CA), as described (19 (link)). Briefly, tissue samples were washed with lysis solution and lysed by probe sonication (Branson SFX150, Branson, Danbury, CT) followed by water bath sonication for 3 min and centrifugation at 4500 × g for 4 min at 4°C. After measuring protein concentration using a Pierce bicinchoninic acid assay (Thermo Fisher Scientific), cytokine and chemokine profiles were determined for each ileum sample (in duplicate) by a Bio-Plex Pro Luminex assay.
3
Protein Analysis of Stem Cell Markers
4
Brain Region Tissue Extraction and Protein Analysis
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Adult male rats (n = 7–8/group) were lethally anesthetized (100 mg/kg pentobarbital, i.p.; Akorn Pharmaceuticals, Lake Forest, IL), and their brains rapidly harvested and frozen on dry ice. The NAc, Hipp, mPFC and CPu were isolated by gross dissection from 2 mm thick sections, collected according to the surface and deep anatomical landmarks illustrated in the Rat Brain Atlas of Paxinos and Watson (1998) . Tissue samples were lysed by manual homogenization in tissue protein extraction reagent (T-PER) supplemented with protease and phosphatase inhibitors (Thermo Scientific, Waltham, MA), using a teflon-coated pestle in hand-held glass homogenizers. Sampling of Hipp and mPFC tissues was done bilaterally, while NAc samples were collected from the left hemisphere only. Lysates were centrifuged at 10,000 × g for 10 min and the cleared lysate subjected to protein quantitation using the Pierce bicinchoninic acid assay (ThermoFisher Scientific, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard. Protein samples were further subjected to SDS-PAGE and Western blotting as described below.
5
Quantification of YBX1, p53, p21, and MDM2 in siRNA Knockdown Cells
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After 96 h, cells transfected with 5 nM Control or YBX1 siRNA were collected and protein isolated by lysis in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (ThermoFisher Scientific). Protein concentration was measured using a Pierce bicinchoninic acid assay (ThermoFisher Scientific). Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and blotted onto PVDF or nitrocellulose membranes. Immunodetection was performed using specific antibodies (monoclonal rabbit anti-YB-1 (ab12148, Abcam; 1:1000), rabbit monoclonal anti-p53 (2527S, Cell Signaling Technology, Danvers, MA, USA; 1:1000), monoclonal rabbit anti-p21 (2947S, Cell Signaling Technology; 1:1000), and mouse anti-MDM2 (ab16895, Abcam, Cambridge, UK; 1:200) and Bio-Rad Clarity (Hercules, CA, USA) enhanced chemiluminescence (ECL). Membranes were then exposed to film or signal images captured using a Bio-Rad ChemiDoc XRS+ System. β-actin (antibody A2228, Sigma Aldrich, St. Louis, MO, USA; 1:1000) or GAPDH (G9545, Sigma Aldrich, 1:5000) served as loading controls. Densitometry was performed using Fiji [67 (link)] or Bio-Rad Image Lab software and a minimum of three biological repeats were performed.
6
Multiplex Cytokine Profiling in Murine Ileitis
7
Western Blot Analysis of Angiogenic Factors
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Protein extraction was performed with ice‐cold RIPA buffer (Thermo) supplemented with a protease inhibitor cocktail followed by homogenization on ice and centrifugation at 14,000g for 15 minutes. Protein concentration in the supernatant was determined with Pierce bicinchoninic acid assay (Thermo). Laemmli buffer (Bio‐Rad) was prepared by adding 5% 2‐mercaptoethanol and used to dilute samples to 50 µg for loading. Proteins were denatured at 95°C for 5 minutes, and separated on Mini‐PROTEAN TGX Stain‐Free 4%–15% gradient gels (Bio‐Rad) for 30 minutes at 300 V, and transferred to nitrocellulose membranes with Trans‐Blot Turbo (Bio‐Rad).
Membranes were blocked for 30 minutes in 10% nonfat dry milk (NFDM) dissolved in TBST (Tris‐buffered saline + 0.1% Tween 20). A CD31 antibody (Abbiotec, San Diego, CA) or a VEGF‐A antibody (Abcam, ab46153, 1:500) was diluted 1:200 in 5% NFDM/TBST and incubated overnight at 4°C with gentle rocking. Membranes were washed (TBST), incubated with horseradish peroxidase goat anti‐Rabbit IgG antibody (1:3,000 in 5% NFDM/TBST) for 1 hour at RT, and then washed again. For visualization, membranes were activated and imaged using the ChemiDoc MP imaging system (Bio‐Rad). As opposed to the use of loading controls, stain‐Free total protein was used for normalization44 , 45 , 46 .
Membranes were blocked for 30 minutes in 10% nonfat dry milk (NFDM) dissolved in TBST (Tris‐buffered saline + 0.1% Tween 20). A CD31 antibody (Abbiotec, San Diego, CA) or a VEGF‐A antibody (Abcam, ab46153, 1:500) was diluted 1:200 in 5% NFDM/TBST and incubated overnight at 4°C with gentle rocking. Membranes were washed (TBST), incubated with horseradish peroxidase goat anti‐Rabbit IgG antibody (1:3,000 in 5% NFDM/TBST) for 1 hour at RT, and then washed again. For visualization, membranes were activated and imaged using the ChemiDoc MP imaging system (Bio‐Rad). As opposed to the use of loading controls, stain‐Free total protein was used for normalization
8
Protein Redox State Monitoring Protocol
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Frozen hippocampus brain tissues were homogenized in a 4 volume of the 10% trichloroacetic acid (VWR International, Radnor, PA, USA) solution using a Kontes glass tissue grinder. Homogenized tissues were incubated on ice for 30 min and centrifuged for 4 min at 8000 rpm at 4 °C in an accuSpin Micro R centrifuge (ThermoFisher Scientific, Waltham, MA, USA). The resultant pellets were then washed with acetone three times, then with ethanol once. Pellets were air-dried, resuspended in the Lysate Buffer (supplied in the -SulfoBiotics- Protein Redox State Monitoring Kit Plus, Dojindo Molecular Technologies Inc., Rockville, MD, USA), supplemented with phenylmethylsulfonyl fluoride (2 mM), leupeptin (10 µg/mL), and aprotinin (10 µg/mL) (MilliporeSigma, Burlington, MA, USA), and sonicated on ice using an ultrasonic processor. Samples were then centrifuged at 13,000 rpm for 10 min, and the supernatants were collected. Protein concentrations of cell lysates were measured using the Pierce bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA, USA).
9
Quantifying ATP Levels in Synchronized Worms
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ATP levels were measured using the CellTiter-Glo Luminescent Cell Viability Assay (Promega G7572), and normalized to protein content determined by the Pierce bicinchoninic acid assay (Thermo Scientific, Rockford, IL) [74 ]. Briefly, 200 synchronized L4 worms were collected, flash frozen and stored at -80 °C. At the time of analysis, worm samples were boiled at 95 °C for 15 min and centrifuged to remove cellular debris. Aliquots of the worm extractions were used for ATP quantification and total protein determination. ATP content was normalized to total protein. In total, four biological replicates were performed.
10
Myc-EXOSC3 Protein Interactome
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N2A cells were transfected with pcDNA3 plasmids containing 2× myc-EXOSC3 or pcDNA3 empty vector (Addgene) using Lipofectamine 2000 (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) as recommended by the manufacturer. Cell pellets were resuspended and lysed in 20 mM Hepes pH 7.2, 100 mM NaCl, and 0.2% Triton X-100. Cell lysate was cleared with centrifugation for 10 min at 10,000 RPM at 4 °C. Protein concentrations were determined using a standard Pierce bicinchoninic acid assay (Thermo Fisher Scientific, 23225). Total lysate (30 μg) was collected for input. For each sample, 20 μl Pierce Anti-c-Myc magnetic beads (Thermo Fisher Scientific #88842) were added to 600 μl to 700 μl of lysate and incubated at room temperature for 1 h while tumbling end over end. Beads were washed 3× and submitted for LC-MS/MS.
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