Cells were lysed on ice for 20 minutes in RIPA buffer (50mM Tris HCl pH 7.4, 150 mM NaCl, 0.1 % (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100) plus complete protease phosphatase inhibitor cocktail (A32961, Thermo Fisher Scientific). Cellular debris was pelleted for 15 minutes at 4 °C and protein concentration was quantitated with the Coomassie (Bradford) Protein Assay (ThermoFisher Scientific). A total of 15μg (organoids) or 30μg (cell lines) of protein lysates were separated on Tris-Glycine (TGX) precast gels (BIO RAD) and transferred to nitrocellulose membranes (Thermo Fisher Scientific). Membranes were probed overnight with antibodies to FoxA1 (1:1000, Abcam 23738), FoxA2 (1:1000, Abcam 108422), NKX2-1 (1:2000, Abcam 133638), CC3 (CST 9664S 1:1000), and Vinculin (1:20000, Abcam 129002). The next day membranes were probed with IRDye 800CW Goat anti-Rabbit IgG Secondary Antibody (1:20000, LI-COR) and imaged with a LI-COR Odyssey CLx and Image Studio Software.
Coomassie bradford protein assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Coomassie (Bradford) Protein Assay is a colorimetric assay used to quantify the total protein concentration in a sample. It is based on the binding of the Coomassie Brilliant Blue G-250 dye to proteins, which results in a color change that can be measured spectrophotometrically.
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Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti rabbit igg secondary antibody, by LI COR (1 mentions)
Complete protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Tgx precast gel, by Bio-Rad (1 mentions)
Varioskan lux, by Thermo Fisher Scientific (1 mentions)
Multiskan fc, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Boston BioProducts (1 mentions)
Paxillin, by Thermo Fisher Scientific (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Clarity enhanced chemiluminescence, by Bio-Rad (1 mentions)
Vcx 130 ultrasonic processor, by Sonics (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue, by Qiagen (1 mentions)
Khb3441, by Thermo Fisher Scientific (1 mentions)
8 protocols using coomassie bradford protein assay
1
Western Blot Analysis of Transcription Factors
2
Endophilin A1 Protein Expression and Labeling
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Full-length rat endophilin A1 (with C108S, E241C, C294S, C295S mutations), its N-BAR domain (with C108S, E241C mutations) were expressed, purified, and labeled with Alexa 594-maleimide as described elsewhere (60 (link)). Briefly, proteins were expressed as GST fusions and purified using GST-affinity chromatography. GST tags were cleaved using PreScission protease, and the BAR proteins were further purified by anion exchange and size-exclusion chromatography techniques. Labeling at the cysteine residue positions was achieved by incubating the protein with Alexa 594-maleimide overnight at 4 °C.
The SH3 domain of rat endophilin A1 was also expressed as GST-fusion protein using a plasmid generously provided by Volker Haucke’s lab. The GST-tag was cleaved using thrombin protease, and the SH3 domain was further purified by anion exchange and size-exclusion chromatography techniques. The purified SH3 domain was labeled at the N terminus using Alexa 594-NHS ester following the same protocol used for N-terminal labeling of TIL as described in the previous section.
Protein concentrations were determined by measuring the absorbance at 280 nm (ɛ280 17,545 M−1 cm−1) for unlabeled full-length endophilin. For all the other cases, the Coomassie (Bradford) Protein Assay (Thermo Scientific, USA) was used for determining protein concentrations.
The SH3 domain of rat endophilin A1 was also expressed as GST-fusion protein using a plasmid generously provided by Volker Haucke’s lab. The GST-tag was cleaved using thrombin protease, and the SH3 domain was further purified by anion exchange and size-exclusion chromatography techniques. The purified SH3 domain was labeled at the N terminus using Alexa 594-NHS ester following the same protocol used for N-terminal labeling of TIL as described in the previous section.
Protein concentrations were determined by measuring the absorbance at 280 nm (ɛ280 17,545 M−1 cm−1) for unlabeled full-length endophilin. For all the other cases, the Coomassie (Bradford) Protein Assay (Thermo Scientific, USA) was used for determining protein concentrations.
3
Quantifying Collagen and Ligand Coupling Efficiency
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Ligand coupling efficiency was determined by measuring how much of the ligand remained in the solution after the coupling process.
The concentration of collagen was quantified using the Bradford protein assay. The Coomassie (Bradford) Protein Assay (Thermo Scientific) reagent was modified to contain 0.035 mg mL−1 sodium docecyl sulphate (SDS),[64 ] in order to increase the steepness of the collagen standard curve and help resolve smaller differences. First, the test solution (5 µL) was added to the assay reagent (250 µL), and the mixture was vortexed and left at room temperature for 10 min. Then, the mixture was vortexed again, before a portion of it (100 µL) was taken out and added to a 96‐well plate. Finally, the absorbance was measured at 570 nm using a microplate reader (Multiskan FC, Thermo Scientific).
The anti‐GLAST antibody used in this study was tagged with either phycoerythrin (PE) or allophycocyanin (APC). For ligand coupling efficiency measurements, anti‐GLAST‐PE was used, and its concentration was estimated by measuring the absorbance at 560 nm using a microplate reader (Varioskan LUX, Thermo Scientific) and comparing the reading to a standard curve.
The concentration of collagen was quantified using the Bradford protein assay. The Coomassie (Bradford) Protein Assay (Thermo Scientific) reagent was modified to contain 0.035 mg mL−1 sodium docecyl sulphate (SDS),[
The anti‐GLAST antibody used in this study was tagged with either phycoerythrin (PE) or allophycocyanin (APC). For ligand coupling efficiency measurements, anti‐GLAST‐PE was used, and its concentration was estimated by measuring the absorbance at 560 nm using a microplate reader (Varioskan LUX, Thermo Scientific) and comparing the reading to a standard curve.
4
Protein Expression Analysis in Cells
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Cells were rinsed with PBS and scraped in ice-cold RIPA lysis buffer (Boston BioProducts) and centrifuged at 8,000 rpm for 5 min at 4 °C. A Coomassie Bradford protein assay (Thermo Fisher) was performed and equal amounts of protein were combined with 4x reducing Laemmli sample buffer (Boston Bioproducts) prior to loading on to SDS-page gel. 20 μg of protein was loaded for each sample and separated by SDS–PAGE, and immunoblotted with antibodies to DRP1 (Novus Biologicals), TRAP1, MFN2, MCT4, VDAC1 (Santa Cruz Biotechnology) NF2, phospho-NF2 (Cell Signaling Technology), FAK (Millipore), Paxillin (Invitrogen) Actin (Sigma). After incubation with peroxidase-conjugated secondary antibodies, the signals were visualized by Clarity™ enhanced chemiluminescence (Bio-Rad) according to the manufacturer’s instruction. Protein band intensity was quantified by ImageJ software.
5
Arsenic Tolerance in Exiguobacterium
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The three Exiguobacterium strains were grown in LB at 25°C with constant agitation (150 rpm) overnight and this culture was used as inoculum to three flasks for each strain: 50 ml of LB as control, 50 ml of LB-As(III), and 50 ml of LB-As(V). Flasks were cultured at 25°C, 150 rpm up to mid-exponential growth phase (OD600 0.4). Arsenic conditions were particular to each strain, half of the MIC value was used as As(III) and As(V) treatment conditions. Bacterial cells were harvested by centrifugation (3,000 g, 10 min) and washed twice with tris–HCl buffer (50 mM, pH 8.5). Bacterial pellets were resuspended in 1 ml of the same buffer supplemented with 1 mM of PMSF (Phenylmethylsulfonyl Fluoride) and were sonicated at 40% amplitude (130 watts, 20 kHz) during 5 min (10 s on and 10 s off cycles) Ultrasonic Processor VCX-130, (Sonics, Inc.). The lysates were centrifuged at 24,000 g for 40 min at 4°C, the supernatants were recovered and stored at −80°C until use. Three independent assays for each strain and condition were performed. Protein concentrations were measured using the Coomassie (Bradford) Protein Assay (Thermo Fisher Scientific).
6
Glucose-Responsive Glucagon Formulation
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To evaluate the glucose responsiveness of the glucagon formulation, ≈10 mg prewashed or washed samples were incubated in 1 mL of PBS solution (pH 7.4) with various glucose concentrations (50, 100, 200, and 400 mg dL−1) at 37 °C with gentle shaking (150 rpm). At predetermined time points, 10 µL of the supernatant was collected, and the released insulin or glucagon was quantified using a Coomassie (Bradford) protein assay (Thermo Fisher Scientific) in a 96 well plate (100 µL well−1). The absorbance was detected at 595 nm on the Infinite 200 Pro multimode plate reader (Tecan Group), and the concentration was calculated with glucagon (8–1000 µg mL−1) standard. Pulsatile release of glucagon was measured using the same procedure by alternating the glucose concentrations between 50 and 400 mg dL−1 in the PBS solution every 30 min of incubation.
7
Quantifying Decellularized Uterine ECM
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All biopsies (n = 8 per group and analysis) for the quantifications were thawed from −20°C and placed on dry filter paper to remove excess fluid and weighed. For DNA quantification, biopsies were independently homogenised in an ATL Buffer (including proteinase K) with a steel bead in a TissueLyser (Qiagen, Sollentuna, Sweden). Qiagen’s DNA extraction kit DNeasy Blood & Tissue was used following the manufacturer’s protocol. The DNA was eluded in 30 μl, and the concentration was established using a NanoDrop (Thermo Scientific, Wilmington, USA). The leftover DNA from five samples per decellularised group, including the DNA extraction from one normal uterus, was then loaded on a 2% agarose gel and run together with a DNA ladder to evaluate the size of the remaining DNA fragments in the decellularised uterine tissues.
Total protein quantification was conducted by Bradford Coomassie protein assay (#1856209; Thermo Scientific) according to standard protocols. ECM macromolecule composition of the decellularised sheep uterine tissue was evaluated using the colourimetric-based assays developed by Biocolor (Carrickfergus, UK) by following the manufacturer’s instructions. To quantify soluble and insoluble collagen, the Sircol S1000 and S2000 were used, respectively. Elastin was assessed by Fastin F2000 Elastin Assay, and sGAGs were quantified using Blyscan™ B1000.
Total protein quantification was conducted by Bradford Coomassie protein assay (#1856209; Thermo Scientific) according to standard protocols. ECM macromolecule composition of the decellularised sheep uterine tissue was evaluated using the colourimetric-based assays developed by Biocolor (Carrickfergus, UK) by following the manufacturer’s instructions. To quantify soluble and insoluble collagen, the Sircol S1000 and S2000 were used, respectively. Elastin was assessed by Fastin F2000 Elastin Assay, and sGAGs were quantified using Blyscan™ B1000.
8
Brain Aβ Protein Fractionation and Quantification
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