The concentration of focal adhesion proteins (vinculin and talin) was determined using the enzyme-linked immunosorbent assay (ELISA). For ELISA measurement Saos-2 were seeded as described in the section cell seeding. To standardize the obtained concentration of the focal adhesion proteins, the amount of total protein in the cells was determined using Quant-iT protein assay kit (Thermo Fisher Scientific) according to the manufacturer's instructions, and the concentration of the focal adhesion proteins was related to the mg of total protein contained in the cells. The cells were harvested after 24 h of culture, suspension was centrifuged and frozen in PBS. The cell homogenates were prepared by ultrasonication for 30 s (amplitude 70%, 24 kHz) using the ultrasonic homogenizer (model 300 V/T, BioLogics, Inc., USA) on ice. The concentration of vinculin and talin in the cell homogenates was determined using the FineTest ELISA kits (FineTest, China) as per manufacturer's instructions. The absorbance was read using a Synergy HT microplate reader (BioTek, USA).
Quant it protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, France, Italy
The Quant-iT Protein Assay Kit is a fluorescence-based assay designed for the quantitation of protein concentration. The kit provides a sensitive and accurate method for measuring protein levels in solution.
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Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (15 mentions)
Qubit fluorimeter, by Thermo Fisher Scientific (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
Glutathione assay kit, by Merck Group (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Oxiselect protein carbonyl elisa kit, by Cell Biolabs (2 mentions)
Gemini xps dual scanning microplate spectrofluorometer, by Molecular Devices (2 mentions)
Softmax pro software, by Molecular Devices (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (2 mentions)
0.45 μm filter, by Sartorius (2 mentions)
Shim pack clc ods, by Shimadzu (2 mentions)
Asys uv340, by Harvard Bioscience (2 mentions)
T9026, by Merck Group (2 mentions)
Pro prep, by iNtRON Biotechnology (2 mentions)
Synergy ht microplate reader, by Agilent Technologies (1 mentions)
Qubit assay tube, by Thermo Fisher Scientific (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by GE Healthcare (1 mentions)
77 protocols using quant it protein assay kit
1
Quantification of Focal Adhesion Proteins
2
Protein Quantitation Using Qubit Fluorometer
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The protein quantitation was carried out using a commercial supply (Quant-iT™ Protein Assay Kit, Thermo Scientific, Waltham, MA, USA). The Quant-iT protein reagent was diluted with a ratio of 1:200 with Quant-iT protein buffer as a working solution. The protein standard solution was prepared by adding 10 μL of BSA standard to 190 μL of working solution to form a final analytic volume of 200 μL. Similarly, the sample solutions were prepared using 180–199 μL of working solution with 1–20 μL of unknown samples to form a final analytic volume of 200 μL. The standard solutions and sample solutions were loaded into the Qubit® assay tubes (Thermo Scientific, Waltham, MA, USA), the assay was performed at room temperature for 15 min, and then the fluorescence reading was measured using a Qubit® fluorometer. Duplicates of the standards and the unknown samples were performed.
3
Cytokine Profiling of Colonic Tissues
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Colonic ex vivo biopsies were cut longitudinally and washed in PBS. MLN, spleen, or strips of ~1 cm2 colonic tissue were placed in 24-flat-bottom well-culture plates (Nunc, Germany) containing 500 μL serum-free RPMI 1,640 medium (Gibco, life technologies, UK) supplemented with penicillin (100 U/mL) and streptomycin (100 μg/mL; PAA Laboratories, Germany). After 18 h at 37°C, culture supernatants were tested for IFN-γ, TNF, MCP-1, and IL-6 by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Biosciences, Germany) on a BD FACSCanto II flow cytometer (BD Biosciences). Nitric oxide (NO) was measured by Griess reaction as described earlier (Heimesaat et al., 2006 (link)), whereas protein concentrations were determined with the Quant-iT™ Protein Assay Kit (Thermo Fisher Scientific, Germany) according to the manufacturer's instructions.
4
Western Blot Analysis of Stress Markers
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The harvested cells were washed once with PBS, lysed with RIPA buffer containing protease inhibitors on ice for 20 min, and centrifuged at 14,000×g for 10 min. The supernatant was collected, and the Quant-iT™ protein assay kit was used to determine protein concentrations (Thermo Fisher Scientific). After boiling an aliquot of the lysate (40 µg protein) for 5 min, the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on their molecular weight. The separated proteins were transferred onto nitrocellulose membranes, which were subsequently incubated with primary antibodies against GRP78, phospho-PERK, phospho-eIF2α, phospho-IRE1, XBP-1, caspase-12, CHOP, and actin, followed by a horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL, USA). The membranes were incubated with enhanced chemiluminescence detection reagents (Amersham, Little Chalfont, Buckinghamshire, UK) and exposed to X-ray film in the dark to visualize protein bands.
5
Protein Extraction and Immunoblotting Protocol
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Drosophila adult heads or brains were homogenized in lysis buffer 1× (10 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10% glycerol, 50 mM NaF, 5 mM DTT, 4 M urea, pH 7.4, plus protease inhibitors and protein content quantified with Quant-iT Protein Assay Kit (#Q33211 Thermo Fisher Scientific). SH-SY5Y cells were homogenized in RIPA buffer (NaCl 150 mM, NP-40 1%, sodium deoxycholate 0.5%, SDS 0.1%, EDTA 2 mM, Tris 50 mM, pH 8.0) added to protease inhibitors, and protein lysates were quantified by Pierce™ BCA Protein Assay Kit (#23225, Thermo Fisher Scientific). Lysates were separated on SDS-PAGE and wet-transferred to nitrocellulose membranes (#NBA083C, Whatman). The primary antibody used were anti-Env (1:100 gifted by Prof. Christophe Terzian), anti-Dcr-2 (1:300 #ab4732, Abcam), anti-h-Dicer (1:3000, #PA5-78446, Thermo Fisher Scientific), anti-hTDP (1:4000, #12892-1-AP, ProteinTech), anti-GFP (1:3000, #A11122, Thermo Fisher Scientific), anti-TBPH (1:4000, homemade, [17 (link)], anti-GAPDH (1:1000 #sc-25778, Santa Cruz), anti-tubulin (1:2000, #CP06, Calbiochem), anti-actin (1:3000, Sigma).
6
Protein Carbonylation Measurement
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Total protein from the cells was isolated using protein lysis buffer, and the protein concentration was measured with a Quant-iT™ protein assay kit (Thermo Fisher Scientific). Protein carbonylation was determined according to the instructions of the OxiSelect™ protein carbonyl ELISA kit (Cell Biolabs, San Diego, CA, USA).
7
Protein Extraction from Frozen Tissue
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From 150 mg of fresh frozen (−80 °C) tissue, total soluble proteins were extracted by homogenization in 3 mL 0.1 M sodium borate buffer (SBB) pH 8.8 containing 10 mM β-mercaptoethanol as described by Hano et al. [46 (link)]. Protein concentration was measured with the Quant-iT Protein Assay Kit and Qubit® 3.0 fluorometer according to manufacturer instructions (Thermo Scientific, Courtaboeuf, France).
8
Purification and Characterization of Plant Cadmium-binding Proteins
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The coding sequences of full-length AdPCS1, AdPCS2, and AdPCS3 proteins were amplified from cDNA using primers listed in Supplementary Table S1 and cloned into the expression vector pET28b in-frame with an N-terminal 6xHis-tag. The expression plasmids were transformed into Escherichia coli Rosetta (DE3) cells, which were induced with 0.5 mM isopropyl ß-D-thiogalactopyranoside and cultured overnight at room temperature. The cells were collected by centrifugation and the soluble fraction of recombinant protein was purified as previously described (Pilati et al., 2014 (link)), quantified with the Quant-iT Protein Assay Kit (Thermo Fisher Scientific), and verified on 10% SDS-PAGE. The PCS activity assay was performed in 100 µl reaction buffer (200 mM HEPES-NaOH pH 8.0, 10 mM β-mercaptoethanol, 12.5 mM GSH, and 100 μM CdSO4) containing recombinant protein (500 ng ml−1) at 35 °C for 60 min, then stopped by adding 25 µl 10% (v/v) trifluoroacetic acid and immediately analyzed for production of PCs with high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC-ESI-MS-MS; Bellini et al., 2019 (link)).
9
Viral Membrane Lysis and Protein Digestion
10
In Vitro Insulin Proteolysis Assay
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A SIF solution with 1% (w/v) pancreatin (4 × USP) was aliquoted and centrifuged for 30 min at 14,000 rpm at 4 °C. Proteolysis was carried out using a water bath on a hot plate at 37 °C. A beaker containing 25 mL SIF with 3% (v/v) of the pancreatin working solution (SIF+P) was placed in the bath and stirred at 200 rpm using a paddle stirrer. Once the temperature reached 37 °C, dry insulin-loaded beads were added to the beaker. A sample was immediately withdrawn and replaced with SIF+P to keep enzyme concentrations constant. Samples were withdrawn at 0, 15, 30, 60, 120, 180, and 240 min and added to 150 µL cold HCl (0.1 M) to stop proteolysis. Samples were then filtered using centrifuge filter tubes, stored at −20 °C and analysed by RP-HPLC. The same protocol was carried out with insulin beads in the absence of pancreatin (negative control), while native insulin solution (1 mg/mL) in the presence of SIF+P was used to confirm enzyme activity [25 (link)]. A protein assay was carried out on the SIF+P solution to standardise the pancreatin concentration from experiment to experiment. This was performed using a Quant-iT™ protein assay kit and according to the manufacturer’s instructions (Thermo Fisher Scientific, Dublin, Ireland). The average pancreatin concentration was 60 ± 6 μg/mL.
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