The protein contents of tissues or cell lysates were measured using a bicinchoninic acid kit (GE Healthcare UK Ltd, Little Chalfont, UK). A suitable amount of total protein of each sample (10–20 µg) was then separated by 10% SDS-PAGE. Following vertical electrophoretic separation, proteins were transferred to polyvinylidene fluoride (PVDF) membranes by wet transfer. Subsequently, PVDF membranes were blocked with 5% fat-free milk for 1 hour at room temperature and then incubated with rabbit anti-UROC28 antibodies, rabbit anti-ZO-1 antibodies, rabbit anti-E-cadherin antibodies, rabbit anti-fibronectin antibodies, rabbit anti-N-cadherin antibodies, and rabbit anti-GAPDH antibodies (Santa Cruz Biotechnology Inc., Dallas, TX, USA) at 4°C overnight.31 (link) Membranes were washed three times in tris-buffered saline with Tween-20 (TBST) and were then incubated with HRP-conjugated secondary antibody (Bio-Rad Laboratories Inc., Hercules, CA, USA) at room temperature for 1 hour. After washing again three times with TBST, exposure and development were performed using an enhanced chemiluminescence (ECL) detection kit (GE Healthcare UK Ltd) and densities were measured using the GS-900™ Calibrated Densitometer with the Image Lab 4.1 Software from Bio-Rad Laboratories Inc. All were performed in triplicate.
Gs 900 calibrated densitometer
Manufactured by Bio-Rad
Sourced in United States
The GS-900 Calibrated Densitometer is a laboratory instrument designed for quantitative analysis of 1D and 2D electrophoresis gels and blots. It measures the optical density of stained sample bands or spots, providing accurate and reproducible quantification.
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Lab products found in correlation
Fast silver stain kit, by Beyotime (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pdquest software, by Bio-Rad (2 mentions)
Tpck trypsin sequencing grade, by Boehringer Ingelheim (2 mentions)
Fujix bas 1000, by Fujifilm (2 mentions)
20 cm tlc plate, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Rabbit anti gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Protein assay kit 2, by Bio-Rad (1 mentions)
Pdquest 2 de 8, by Bio-Rad (1 mentions)
Bradford reagent, by Bio-Rad (1 mentions)
Readyprep protein extraction kit, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
21 protocols using gs 900 calibrated densitometer
1
Western Blot Analysis of Cell Proteins
2
Protein extraction and Western blotting of SGBS cells
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At day nine of differentiation SGBS cells were homogenized in ice cold M-PER Mammalian Protein Extraction Reagent (#78501, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 250 mM Sucrose, 1 mM EDTA, 1% NP-40 substitute and complete protease inhibitor cocktail (Roche GmbH, Mannheim, Germany). After incubation on ice for 10 min and centrifugation at 10,000 × g for 10 min at 4 °C, the middle phase was removed and proteins were quantified using the Bio-Rad DC Protein Assay Kit. SDS-PAGE followed by Western blotting was performed using antibodies against FAM46A (1:1000 dilution, PA5-23898, Thermo Fisher Scientific, Waltham, MA, USA) or HSL (1:2000 dilution, #4107, Cell Signalling Technology, Danvers, MA, USA) following manufacturer’s standard protocol and ß-Actin (1:4000 dilution, #3700, Cell Signalling Technology, Danvers, MA, USA) was used for normalization. Species appropriate secondary antibodies conjugated to horseradish peroxidase (1:10,000 dilution, anti-mouse 711-035-152 or anti rabbit 715-035-150, Jackson ImmunoResearch Laboratories, Inc, West Grove, PA, USA) were used and proteins visualized and quantified by enhanced chemiluminescence using the Bio-Rad GS-900 calibrated Densitometer.
3
Quantitative Protein Estimation via Solid-Phase Assay
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Estimation of total protein concentration was carried out using a solid-phase protein assay as previously described (Noaman and Coorssen, 2018 (link)). Briefly, solubilized serum samples were serially diluted to yield concentrations appropriate for measurement against a linear calibration curve (0.5–0.05 mg/ml of BSA in 2DE lysis buffer). 1 μl of each dilution was dot-blotted in triplicate onto WhatmanTM 3MM chromatography paper (GE Healthcare, Chicago, IL, United States). Dried blots were washed with methanol for 5 min, dried under ambient conditions, and stained for 10 min with colloidal CBB (cCBB). Destaining was carried out for 5 min × 5 min with ddH2O, and blots were dried prior to imaging via reflective densitometry using the GS-900 Calibrated Densitometer (Bio-Rad, Hercules, CA, United States) followed by quantitation using ImageLab (Bio-Rad, Hercules, CA, United States) and Microsoft Excel 2013.
4
2D-PAGE Profiling of Root Proteome
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2D-PAGE was performed according to the method described by Valledor et al. [75 (link)], with minor modifications. The root proteins were solubilized in lysis solution and proteins concentration was determined using Bio-Rad Protein Assay Kit II (Bio-Rad, Shanghai, China), with Bovine serum albumin (BSA) as a standard protein. About 900 μg of protein was separated on a 17 cm pH 4–7 linear IPG strip (Bio-Rad) and actively rehydrated at 50 V for 14 h at 20 °C. Subsequently, focusing was performed under following conditions: 250 V for 1 h, 500 V for 1 h, 1000 V for 1 h, 8000 V for 4 h, and 8000 V to achieve 80,000 V-h. Strips were immediately equilibrated twices. The Second-dimension electrophoresis was performed on 12% polyacrylamide gels. Gels were stained with Coomassie Brilliant Blue (CBB) G-250. Each sample was run in 3 independent biological replicates.
Gels were visualized using a GS-900 Calibrated Densitometer (Bio-Rad, Taiwan, China) at a resolution of 600 dpi. Images were analyzed using the analytical software PDQuest 2-DE 8.0.1 (Bio-Rad, Hercules, CA, USA) for spot detection, gel matching, and statistical analysis of spots. The selection of protein spots of interest for analysis by MS was based on a fold change ≥1.5 (p < 0.05).
Gels were visualized using a GS-900 Calibrated Densitometer (Bio-Rad, Taiwan, China) at a resolution of 600 dpi. Images were analyzed using the analytical software PDQuest 2-DE 8.0.1 (Bio-Rad, Hercules, CA, USA) for spot detection, gel matching, and statistical analysis of spots. The selection of protein spots of interest for analysis by MS was based on a fold change ≥1.5 (p < 0.05).
5
Western Blot Protein Extraction and Analysis
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Cells or mouse tissues were lysed in Tissue Protein Extraction Reagent (T-PER, Thermo Fisher) supplemented with EDTA-free protease and phosphatase inhibitory cocktails. Homogenates were freeze-thawed three times in liquid nitrogen and clarified by centrifugation at 11,000 ×g for 30 min at 4°C. Protein concentration was determined using Bradford reagent (Bio-Rad protein assay, Bio-Rad). The resulting protein extracts were fractionated on SDS-12% PAGE and transferred onto PVDF or nitrocellulose membranes for immunoblot analysis. Membranes were blocked with 5% nonfat dried milk in TBS with 1% Tween 20 for 1 h and incubated with the primary antibodies diluted in 3% BSA in TBS overnight at 4°C. The primary antibodies used are rabbit anti-IF1,46 (link) rabbit anti-β-F1,40 (link) mouse anti-α-tubulin, mouse anti-CD4 and rabbit anti-APRT. Peroxidase-conjugated anti-mouse or anti-rabbit IgGs (1/5,000) were diluted in TBS with 1% Tween 20 and used as secondary antibodies. The Novex® ECL system (Thermo Fisher) was used to visualize the bands. The intensity of the bands was quantified using a GS-900™ Calibrated Densitometer (Bio-Rad) and the Analyze Gel command of ImageJ software (NIH).
6
Placental Protein Expression Analysis
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The total protein was extracted from the placenta by ReadyPrep™ Protein Extraction Kit (Bio-Rad, Hercules, CA) and the concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). The protein was separated by SGS-PAGE and it was transferred to the PVDF membrane. Afterwards, the membrane was incubated with primary antibody (anti-GRP78, (78 kDa glucose-regulated protein 78), anti-IRE-1α (inositol-requiring enzyme-1α), anti-p-IRE1α, anti-eIF2α (eukaryotic initiation factor alpha 2), anti-p-eIF2α, and anti-β-actin) for overnight followed by HRP-conjugated secondary antibody. The immunoreactive proteins were detected by Clarity™ Western ECL Blotting Substrates (Bio-Rad, Hercules, CA) and the density was measured by GS-900™ Calibrated Densitometer (Bio-Rad, Hercules, CA).
7
Immunogenic Protein Expression Analysis
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The survival analysis was performed using a comparison of survival curves and log-rank (Mantel–Cox) test for survival assays with the PRISM computer program (GraphPad, San Diego, Calif.). p < 0.05, p < 0.01, or p < 0.001 was considered statistically significant. Protein bands of interest were scanned with the GS-900 calibrated densitometer (Biorad). The ImageLab program was used to establish changes in the densitometry of the Western blot immunogenic bands corresponding to the treatment and the different number of immunizations. Data represent mean percentages ± SD of three independent experiments (bands of three independent repetitions of Western blot). The data obtained were statistically analyzed using t-tests (and nonparametric tests), and then an unpaired t-test. A level of significance with p < 0.05, p < 0.01, or p < 0.001 was considered to establish significant differences between compared immunogenic bands.
8
Native PAGE and Zymography of Protein Activities
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Cell lysates from transfected NSC-34 cells were generated as aforementioned, with the exception that lysis buffer was 100 mM Tris-base (pH 7.5) with 0.1% TX-100 (v/v) and protease inhibitor. Cell lysates were mixed 1:2 with 3× native-PAGE sample buffer (240 mM Tris–HCl [pH 6.8], 30% glycerol [v/v], 0.03% bromophenol blue [w/v]) and loaded into Tris–glycine native-PAGE gels (4.5% stacking gel [pH 8.8], 7.5% resolving gel [pH 8.8]). Samples were electrophoresed for 30 min at 60 V and then for 2.5 h at a constant voltage of 125 V at 4 °C. Following native-PAGE, EGFP, or TdTomato signal in the gel was detected using a ChemiDoc MP Imaging System (Bio-Rad). Gels were then subjected to zymography as described previously (64 (link)) and imaged using a GS-900 Calibrated Densitometer (Bio-Rad). Quantification of fluorescence signal and enzymatic activity was performed using ImageJ (version 1.53c) (88 (link)).
9
Western Blot Analysis of Cartilage Degradation
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A total of 50 μg of proteins from either cell lysate or articular cartilage homogenate were loaded onto a 12% SDS-PAGE gel. After transfer, membranes were blocked by 5% non-fat milk and incubated with different primary antibodies for overnight at 4°C. Next day, corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated. After washing, the membranes were finally carried out with SuperSignal1 West Pico Chemiluminescent Substrate Kit (Thermo Scientific, Waltham, MA, USA). Rabbit anti-MMP1 (1:2,000), rabbit anti-MMP3 (1:2,000), rabbit anti-MMP9 (1:2,000), rabbit anti-MMP13 (1:2,000), rabbit anti-ADAMTS4 (1:2,000), rabbit anti-ADAMTS5 (1:2,000), mouse anti-GAPDH (1:1,000), rabbit anti-IκBa (1:2,000), rabbit anti-p- IκBa (1:2,000), rabbit anti-p65 (1:2,000), rabbit anti-p-p65 (1:2,000), goat anti-mouse immunoglobulin G H&L (HRP) (1:3,000), and goat anti-rabbit immunoglobulin G (HRP) (1:3,000) were purchased from Abcam (Cambridge, MA, USA). The western blot results were quantitated and analyzed using GS-900 Calibrated Densitometer and software Image Lab (Bio-Rad, Hercules, CA, USA) following manufacturer’s instructions.
10
SDS-PAGE Protein Separation and Analysis
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The proteins extracted from each sample (80 μg of BSA protein equivalent) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gel (Laemmli, 1970 (link)), using the Protean II-XL (20 × 20 cm) system from Bio-Rad (Hercules, CA, USA) with a voltage run of 80 V until the dye reached the bottom of the gel. Gels were stained with Coomassie Brilliant Blue R-250 (Neuhoff et al., 1988 (link)), and images were acquired with a GS-900 Calibrated Densitometer from Bio-Rad. Images were analyzed with the software ImageLabTM 5.2.1, also from Bio-Rad, bands being automatically detected and confirmed by visual inspection. The optical density (OD) of each band was normalized against the combination of all bands for each sample. Band molecular masses were calculated by comparing mobilities with those of protein standard markers (SDS Molecular weight standards, Broad range, Bio-Rad).
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