Total cell and tissue lysates were prepared by homogenizing a portion of the tissue directly in RIPA buffer containing Protease Inhibitor Cocktail Tablets (Roche Applied Science, Indianapolis, IN, USA). Serum extracts were diluted in RIPA buffer with the protease inhibitor. Myotube extracts were briefly sonicated using a Model 100 Sonic Dismembrator (Fisher Scientific, Asheville, NC, USA). Cell, tissue and serum extracts were centrifuged at 14,000 g for 10 min at 4°C. Concentrations of lysates were determined by using the Bradford Protein Assay Kit (Fisher Scientific) with bovine serum albumin as standard.
Bradford protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bradford protein assay kit is a colorimetric assay used to quantify the total protein concentration in a sample. It relies on the binding of Coomassie Brilliant Blue dye to proteins, which causes a shift in the dye's absorption spectrum that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (12 mentions)
Protease inhibitor cocktail, by Merck Group (7 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (6 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ecl kit, by Merck Group (5 mentions)
Albumin standard, by Thermo Fisher Scientific (4 mentions)
Lds sample loading buffer, by Thermo Fisher Scientific (4 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (4 mentions)
Fluostar optima plate reader, by BMG Labtech (4 mentions)
Lysis buffer, by Beyotime (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (3 mentions)
Ecl reagent, by Cytiva (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Enhanced chemiluminescence detection reagent, by Cytiva (3 mentions)
Protease and phosphatase inhibitor, by Merck Group (3 mentions)
Polyvinylidene fluoride (pvdf), by GE Healthcare (3 mentions)
Sulfo smcc crosslinker, by Thermo Fisher Scientific (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Goat anti mouse il 33 affinity purified polyclonal antibody, by R&D Systems (2 mentions)
T per tissue extraction buffer, by Thermo Fisher Scientific (2 mentions)
114 protocols using bradford protein assay kit
1
Tissue and Serum Protein Extraction
2
Recombinant MLP Protein Expression in E. coli
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The co-expression of HIS6-TxtAA and HIS6-TxtBA with HIS6-tagged MLPs was conducted as previously described (Li et al., 2019a (link)). Briefly, the expression strain E. coli BL21(DE3)ybdZ:aac(3)IV (Table 1 ) containing either pACYCDuet-1/HIS6-txtAA or pACYCDuet-1/HIS6-txtBA, with and without a pET28b-derived MLP expression plasmid (Table 2 ), was cultured overnight in 3 mL of LB medium supplemented with 1% w/v glucose and the appropriate antibiotics. The overnight cultures were subcultured into fresh LB medium containing appropriate antibiotics, and the cultures were incubated at 37°C and 200 rpm until the OD600 reached 0.4–0.6. The production of the HIS6-tagged proteins was induced by adding 1 mM isopropyl β-d-thiogalactopyranoside (IPTG) and then incubating the cultures at 16°C and 200 rpm for 48 h. Cells from 1 mL of culture were harvested and were resuspended in 200 μL of 50 mM Tris–HCl (pH 8.0) containing 1 × cOmplete EDTA-free protease inhibitor. The cells were lysed by sonication and the cell debris was removed by centrifugation. The soluble proteins were collected, and the protein concentration was quantified using a Bradford protein assay kit (Fisher Scientific).
3
Synthesis and Validation of ADRQβ-004 Vaccine
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Peptide synthesis and vaccine preparation were performed as described previously [22 (link)]. In brief, the epitope CGITEEAGY (termed ADR-004), which belongs to the second extracellular loop (ECL2) of α1D-AR, was synthesized and validated by GL Ltd. (Shanghai, China) with a purity above 98%. VLP was expressed and purified and then identified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy. ADR-004 was covalently conjugated to VLP using a Sulfo-SMCC crosslinker (Thermo Fisher Scientific, Massachusetts, USA) to produce the vaccine called ADRQβ-004. The conjugation rate of the ADRQβ-004 vaccine was analyzed by SDS-PAGE. The concentration of the vaccine was determined using a Bradford protein assay kit (Thermo Fisher Scientific, Massachusetts, USA).
4
Tissue Protein Extraction and Western Blotting
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After weighing, the frozen tissues were homogenized by lysis in ice-cold modified RIPA buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. Then, the homogenates were centrifuged, and the protein concentration of the supernatant was determined using a Bradford protein assay kit (Thermo Fisher Scientific, Massachusetts, USA). Twenty-five micrograms of protein from heart and liver tissue was added to a 15-μL protein loading system, while 50 μg of protein from fat tissue was added to a 20-μL protein loading system. Proteins were separated by 10% PAGE gels (Yamei, Shanghai, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Massachusetts, USA). The blots were probed with the following primary antibodies for a 12-h incubation: anti-tyrosine hydroxylase (TH) monoclonal antibody (1:1000, Santa Cruz Biotechnology, Texas, USA), anti-peroxisome proliferator activated receptor β (PPARβ) (1:1000, Santa Cruz Biotechnology, Texas, USA), and anti-GAPDH monoclonal antibody (1:3000, Proteintech, Wuhan, Hubei). The Western blotting results were quantified using Image Lab software (Bio-Rad Laboratories, CA, USA) and were expressed normalized to the mean of the control group.
5
Western Blot Analysis of Apoptosis Markers
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Cell lysate was boiled in a sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, 20% glycerol, and 10% 2-mercaptoethanol), and protein concentration was determined using a Bradford protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) with bovine serum albumin as the standard [47 (link)]. Following protein transfer, the membrane was blocked with 5% skim milk in PBS Tween- (PBST-) 20 for 2 h at room temperature and then incubated overnight with antibodies at 4°C (the primary antibodies include Bcl2, 1 : 1000, Cell Signaling Technology, #3498; Bax, 1 : 1000, Cell Signaling Technology, #2772; caspase-9, 1 : 1000, Cell Signaling Technology, #9504; c-IAP, 1 : 1000, Cell Signaling Technology, #4952). The membranes were then washed with PBST containing 0.1% Tween. After three washes in PBST, each blot was incubated with peroxidase-conjugated secondary antibody for 1 h at 37°C. Labeled proteins were visualized using the Odyssey infrared scanner (LI-COR, Lincoln, NB, USA) [48 (link)]. Signals were densitometrically assessed and normalized to the β-actin signals, and an enhanced chemiluminescence detection system (Amersham, Piscataway, NJ, USA) was used to visualize the antibody-specific proteins in accordance with the manufacturer's recommended protocol [49 (link)].
6
Antibody Characterization for Signaling Pathways
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Primary antibodies for AKT (total, rabbit), phospho AKT (Ser473, rabbit), glycogen synthase kinase 3 beta (GSK3β; total, rabbit), phospho GSK3β (Ser9, rabbit), and green fluorescent protein (GFP, rabbit) were from Cell Signaling Technology (Danvers, MA, USA). Those for, IP3R1 (rabbit), fetal and adult testis-expressed 1 (FATE-1; mouse), eNOS (NOS3, mouse), and iNOS (NOS2, mouse) were from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies for CypD (cyclophilin F, mouse) and VDAC1 (mouse) were from Abcam (Paris, France) and the one for tubulin α (mouse) was from Sigma-Aldrich (Saint-Quentin Fallavier, France). Duolink® proximity ligation assay and Trizol Reagent kits were from Sigma-Aldrich (Saint-Quentin Fallavier, France). NucleoSpin® DNA RapidLyse kit was from Macherey-Nagel (Hoerdt, France). PrimeScript reagents RT kit (Takara Bio Inc., USA). Bradford protein assay kit was from ThermoFisher Scientific (Courtaboeuf, France). Other chemicals were from Sigma-Aldrich (Saint-Quentin Fallavier, France), Qiagen (Courtaboeuf, France), ThermoFisher Scientific (Courtaboeuf, France), or Cell Signaling Technology (Danvers, MA, USA).
7
Small Extracellular Vesicle Isolation
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Small EVs were isolated from cell culture supernatant by UC, as described previously [6 (link)]. Conditioned medium (240 mL) was harvested after 7 days of incubation and immediately centrifuged at 300× g for 10 min. Apoptotic bodies were removed by centrifugation at 2000× g using a Sigma13190 rotor (MBI) for 20 min. Samples were then spun at 16,500× g for 1 h using an SW28 Ti rotor (Beckman Coulter, Indianapolis, USA) to deplete microvesicles. To pellet sEVs, the same rotor was used to centrifuge samples for 3 h at 100,000× g. Collected sEVs were washed with PBS and centrifuged at 100,000× g for 1 h and finally resuspended in 200 μL PBS and stored at −80 °C. A Bradford protein assay kit (Thermo Scientific, Rockford, USA, Cat No. 23236) was used to assess sample protein concentrations. The average amount of EV protein from three replicate samples was roughly 22 ± 5, 27 ± 6 and 34 ± 6 μg for MCF10A, MCF7 and MDA-MB-231 cell lines, respectively. These samples were used for proteomic analysis.
8
Quantifying IL-33 in Hpb-infected Intestine
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Duodenal intestinal tissue was collected from Hpb inoculated VillinCre-A2BARfl/fl and control VillinCre mice, and 24 h later, along with naïve controls, was lysed in T-PER tissue extraction buffer (Thermo Scientific, Waltham, MA, catalog#78510), with 2× protease inhibitors and 2× EDTA (Thermo Scientific, catalog# 78444). Protein concentration was measured using Bradford protein assay kit (Thermo Scientific, catalog# 23200). 40ug of total protein was loaded to SDS-PAGE gel. Gel was transferred, and PVDF membrane was probed with 0.4 μg/mL of Goat Anti-Mouse IL-33 affinity-purified polyclonal antibody (R&D Biosystems, catalog # AF3626) overnight at 4°C and followed by HRP-conjugated anti-Goat IgG secondary antibody (R&D Biosystems, catalog # HAF109) for 1 h.
9
Reagents and Chemicals Used in Biological Assays
10
Sotorasib's Impact on RAS Signaling
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H2122 cells (5 × 105 cells per well) were seeded in 6-well plates. The next day, supernatants were replaced with media containing 100 nmol/L sotorasib or DMSO. After 24-hour incubation, whole-cell lysates were generated in RIPA buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% NP-40, and 0.1% SDS), supplemented with protease inhibitors (40 μg/mL PMSF, 2 μg/mL antipain, 2 μg/mL pepstatin A, 20 μg/mL leupeptin, and 20 μg/mL aprotinin), and phosphatase inhibitors (10 mmol/L NaF, 1 mmol/L Na3VO4, 10 mmol/L β-glycerophosphate, and 10 mmol/L sodium pyrophosphate). After clarification of debris by centrifugation, samples were quantified by using the Bradford Protein Assay Kit (Thermo Fisher). Total lysate protein (20 μg) was resolved by SDS-PAGE and transferred onto PVDF membranes (MilliporeSigma). Membranes were incubated with appropriate primary and secondary antibodies labeled with IRDye (680 nm) and visualized using an Odyssey CLx Imaging System (LI-COR). Antibodies used here were monoclonal pan-RAS antibody (1:1,000; clone Ab-3, Millipore), mouse monoclonal ERK2 antibody (1:1,000; clone D2, Santa Cruz Biotechnology), and IRDye 680LT Goat anti-Mouse IgG (H + L; 1:10,000; LI-COR).
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