ROS production was evaluated as previously described [18 (link)]. Briefly, cells were seeded in a 24-well plate at a density of 30,000 cells/well and incubated for 30 min with 5 μM of carboxy-H2DCFDA (Molecular Probes, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), a carboxyl derivative of 2′,7′-dichlorofluorescein, in HEPES buffer. Then, cells were treated with sera or isolated lipoprotein fractions from carriers of LCAT mutations and controls for 1 h. The oxidation of the probe was detected by monitoring fluorescence at 517–527 nm with the Synergy H1 multi-mode reader and the Gen5 software (BioTek, Agilent, Santa Clara, CA, USA). For each sample, fluorescence was normalized by the protein concentration of the total cell lysate, measured by the micro-bicinchoninic acid assay (Thermo Scientific, Waltham, MA, USA).
Micro bicinchoninic acid assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Micro-bicinchoninic acid (Micro-BCA) assay is a colorimetric method for the quantitative determination of protein concentration. It is a sensitive and accurate technique used to measure protein levels in small sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
Synergy h1, by Agilent Technologies (2 mentions)
Exoquick, by System Biosciences (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Carboxy h2dcfda, by Thermo Fisher Scientific (1 mentions)
Intercept tbs blocking buffer, by LI COR (1 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Odyssey system, by LI COR (1 mentions)
Tsq quantiva, by Thermo Fisher Scientific (1 mentions)
Pinpoint 1, by Thermo Fisher Scientific (1 mentions)
Nanoacquity liquid chromatography system, by Waters Corporation (1 mentions)
Octylglucoside, by Anatrace (1 mentions)
3 3 cholamidopropyl dimethylammonio 1 propane sulfonate, by Anatrace (1 mentions)
Imperial protein stain solution, by Thermo Fisher Scientific (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
1 3 bis 1 2 dioleoyl sn glycero 3 phospho glycerol, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phospho 1 rac glycerol, by Avanti Polar Lipids (1 mentions)
1 palmitoyl 2 9 10 dibromo stearoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
L α phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
Cardiolipin, by Avanti Polar Lipids (1 mentions)
12 protocols using micro bicinchoninic acid assay
1
Measuring Intracellular ROS Production
2
Exosome Harvesting and Proteomic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Laboratory investigators (NKG, LMW, JSS, and KMD) were blinded to patient characteristics until completion of all assays and data generation. Exosomes were harvested from human serum via Exoquick (Systems Biosciences, Inc., Mountain View, CA) after filtration through a 0.2 micron filter. Protein content of the purified exosomes was quantitated by micro bicinchoninic acid assay (Thermo Fisher Scientific, Inc. Rockford, IL). Twenty µg of protein of each sample was resolved by SDS-PAGE and processed via digestion. Samples subjected to full proteomic characterization by LC-MS/MS were divided into ten gel fractions prior to digestion [12] (link). Exosome samples for MRM-MS were left unfractionated. All samples were digested with trypsin at a 20:1 ratio (sample:protease), as previously published [12] (link).
3
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were collected in lysis buffer: RIPA buffer (Thermo Scientific, Waltham, MA) containing 1 × protease and phosphatase inhibitor cocktail (78,440, Thermo Fisher Scientific, Waltham, MA). After a brief incubation on ice, the lysates were homogenized by passing the samples through 26-G needles, followed by centrifugation at 16,000 g, 4℃, for 20 min to collect the supernatant. Protein concentration was quantified by microbicinchoninic acid assay (23,227, Thermo Fisher Scientific, Waltham, MA). Lysates (30 µg) were separated by SDS-PAGE under reducing conditions, transferred to nitrocellulose membranes, and blocked in Intercept TBS blocking buffer (927–66,003, LI-COR Biosciences, Lincoln, NE). Membranes were incubated with primary antibodies diluted in blocking buffer overnight at 4 °C, (UGDH 1:1000), (GAPDH 1:10,000), washed with Tris-buffered saline containing 0.1% Tween 20 (TBST), then incubated with fluorescent secondary mouse or rabbit IgG antibodies (IRDye, LI-COR Biosciences, Lincoln, NE). Images were generated using the Odyssey system and software (LI-COR Biosciences, Lincoln, NE).
4
Quantifying HER2 Protein Levels by SRM Mass Spectrometry
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HER2 protein level was quantified by SRM mass spectrometry as previously described [9–11 (link)]. Briefly, one section was cut for hematoxylin and eosin (H&E) staining, and multiple sections were cut on to Director® microdissection slides and stained with eosin. The H&E was used to guide tumor area selection on the slide sections, which were then microdissected (Molecular Machines & Industries, Eching, Germany). Collected tumor tissue was solubilized using Liquid Tissue® according to manufacturer’s instructions. Total protein concentration from each sample was measured by a micro bicinchoninic acid assay (Thermo Fisher Scientific Inc, Waltham, MA).
A mixture of stable isotope-labeled synthetic peptides was added to the liquefied tumor samples as internal standards. All samples were analyzed in triplicate using a triple quadrupole mass spectrometer (TSQ Quantiva™, Thermo Scientific, Waltham, MA) interfacing with a nanoACQUITY liquid chromatography system (Waters Corporation, Milford, MA). A 100 µm inner diameter chromatographic column packed with C18 resin (ProntoSIL 200-5-C18AQ; Bischoff Chromatography, Germany) was used for peptide separation before mass spectrometry analysis. For protein quantitation, peak areas from each endogenous and internal standard peptide were calculated and ratios were determined using PinPoint 1.3 (Thermo Scientific, Waltham, MA).
A mixture of stable isotope-labeled synthetic peptides was added to the liquefied tumor samples as internal standards. All samples were analyzed in triplicate using a triple quadrupole mass spectrometer (TSQ Quantiva™, Thermo Scientific, Waltham, MA) interfacing with a nanoACQUITY liquid chromatography system (Waters Corporation, Milford, MA). A 100 µm inner diameter chromatographic column packed with C18 resin (ProntoSIL 200-5-C18AQ; Bischoff Chromatography, Germany) was used for peptide separation before mass spectrometry analysis. For protein quantitation, peak areas from each endogenous and internal standard peptide were calculated and ratios were determined using PinPoint 1.3 (Thermo Scientific, Waltham, MA).
5
Optimized Membrane Protein Purification and Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All lipids were purchased from Avanti Polar Lipids: L-α-phosphatidylethanolamine (PE) from E. coli, L-α-phosphatidylglycerol (PG) from E. coli, cardiolipin (CL) from E. coli, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-(9,10-dibromo)stearoyl-sn-glycero-3-phosphocholine (16:0–18:0(9-10Br)PC), and 1′,3′-bis[1,2-dioleoyl-sn-glycero-3-phospho]-glycerol (18:1 CL). All detergents were purchased from Anatrace: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), octylglucoside (OG), polyoxyethylene(9)dodecyl ether (C12E9), n-Decyl-α-D-Maltoside (DM), n-Dodecyl-α-D-Maltoside (DDM), lauryldimethylamine-N-Oxide (LDAO). The Imperial Protein Stain solution and micro-bicinchoninic acid assay (BCA) were purchased from Thermo Fisher. The QuikChange Lightning site-directed mutagenesis kit was purchased from Agilent Technologies. 1,5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) and 3-(N-maleimidopropionyl) biocytin (MPB) were purchased from Molecular Probes. 4-Nitrophenyl- β-D-galactopyranoside (NPG) and N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM) were purchased from Sigma Aldrich. β-D-Galactopyranosyl 1-thio-β-D-galactopyranoside (TDG) was obtained from Cayman Chemicals.
6
Fibroblast Proteome Analysis in Fibrosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured HDFs were lysed with radio-immunoprecipitation assay lysis buffer (Beyotime, Jiangsu, China). Protein concentrations were determined using a micro bicinchoninic acid assay (Thermo Fisher Scientific). Total proteins were separated on acrylamide gels, and immunodetected with the primary antibodies that included the following: Col1a2, Col3a1 (Genetex, Biozol, Eching, Germany, 1:1000), α-SMA, Smad6, Smad7, TGF-β receptor I (TGFβRI), TGF-β receptor II (TGFβRII) (Abcam, Cambridge, UK, 1:1000), Smad2, p-Smad2, Smad3, p-Smad3, Smad4, Smad1/5/9, p-Smad1/5/9 (Cell Signaling Technology, Beverly, MA, 1:1000). Immunoreactive bands were quantitatively analyzed with ImageJ software.
7
Extracellular Vesicle Protein Characterization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein concentrations of EV samples were determined using the micro-bicinchoninic acid assay (Thermo Fisher, Waltham, USA). Western blots were performed with 5 μg of the concentrated EV fractions, which were treated with sample buffer (dithiothreitol, 0.1% SDS, 0.1 M Tris HCl; pH 7.0) and boiled for 5 min at 95 °C before separation on 12% SDS-polyacrylamide gel electrophoresis gels. The samples were transferred to polyvinylidene fluoride membranes (Merck Group, Darmstadt, Germany). The membranes were blocked by 5% milk solution (skim milk powder (Thermo Fisher, Waltham, USA) dissolved in Tris-buffered saline solution with 1% Tween-20) for 1 h at RT and stained with antibodies recognizing “exosomal marker” proteins including CD63 (1:500, Biorbyt, Cambridge, UK), TSG101 (1:500, GeneTex, Irvine, USA), Alix (1:500, BD Transduction Laboratories, San Jose, USA), and negative control calnexin (1:500, Abcam, Cambridge, UK) followed by a 1-h incubation with a matched horseradish peroxidase-labeled secondary antibody. Immunoreactivity was detected using chemiluminescence detection kit reagents and a Chemidoc Station (Biorad, Hercules, USA). ImageJ was used for densitometric analysis of each blot. The Western blotting procedures were repeated three times per sample. Please also refer to Supplementary Figure S2 for uncropped Western blots.
8
Keratinocyte Cytokine Expression Analysis
9
EV characterization of NPCs, BM-MSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EV characterization was performed according to the International Society for Extracellular Vesicles guidelines [15] (link), through transmission electron microscopy (TEM), singleparticle analysis (qNano, Izon Science, New Zealand) [16] (link) and assessment of CD63, CD9, TSG101, and lamin A/C expression by Western blot, comparing NPCs, BM-MSCs, and their EVs. EVs were quantified using the micro-bicinchoninic acid assay (Thermo Fisher).
10
Evaluating HDL Antioxidant Effects in Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PNT2, LNCaP and PC-3 cells seeded in 24-well black plates were incubated with HDL, sHDL or apoA-I at 0.5 mg/ml up to 16 h in RPMI 1640 (supplemented with 1% bovine serum albumin for LNCaP). Cells were then washed with PBS and loaded with 3 μM 2′,7′-dichlorofluorescein (Life Technologies, USA) for 30 minutes. After RPMI 1640 replenishment, cells were stimulated or not with 0.5 mM H2O2 for 1 h and ROS production was measured by fluorescence on a Synergy H1 multi-mode reader equipped with the Gen5 software (BioTek, USA). To evaluate the role of AR signalling in ROS generation and HDL antioxidant effect, in separate experiments LNCaP were maintained in RPMI 1640 without phenol red and incubated overnight with the AR antagonist bicalutamide (100 nM, Sigma-Aldrich). To evaluate the role of cell cholesterol content on HDL antioxidant effect, cells were pre-treated overnight with LDL (50 μg/ml) or for 1 h with 2.5 mM beta-methylcyclodextrin (βMCD, Sigma Aldrich, Germany).
For each sample, fluorescence was normalized by the protein concentration of the total cell lysate, measured by the micro-bicinchoninic acid assay (Thermo Scientific, USA).
A standard HDL preparation from pooled plasma was tested in each experiment for inter-assay data correction.
For each sample, fluorescence was normalized by the protein concentration of the total cell lysate, measured by the micro-bicinchoninic acid assay (Thermo Scientific, USA).
A standard HDL preparation from pooled plasma was tested in each experiment for inter-assay data correction.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!