Cell extracts were harvested from the SP and non-SP cells using RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) and protein concentration was determined using a Bradford assay (Sigma-Aldrich) (18 (link)). Protein lysates (40 μg) from each sample were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich). Separated proteins were transferred to a polyvinylidene difluoride membrane (Sigma-Aldrich). The membranes were treated with the primary antibodies against β-catenin (1:2,400; cat. no. ab6302; Abcam, Shanghai, China) and GAPDH (0.7 μg/ml; cat. no. ab37168, Abcam). Subsequently, the membranes were incubated in the secondary antibodies horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG with alkaline phosphatase markers; cat. no. ab97048, Abcam). The protein was detected using chemiluminescence reagents (Amersham Biosciences). Blots were scanned using a Bio-Rad GS-710 densitometer (Bio-Rad Laboratories, Inc.).
Gs710 densitometer
Manufactured by Bio-Rad
Sourced in United States
The GS710 densitometer is a lab equipment product manufactured by Bio-Rad. It is designed to measure the optical density of samples, such as gels or blots, in a quantitative manner. The device captures and analyzes images to provide accurate density measurements for various applications.
Automatically generated - may contain errors
Lab products found in correlation
Image master platinum 5.0 image analysis program, by Cytiva (7 mentions)
Ph 3 10 nonlinear gradient strips, by Cytiva (3 mentions)
Nonlinear gradient strips, by Cytiva (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Chemiluminescence reagent, by Cytiva (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Merck Group (1 mentions)
Bradford assay, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Immobilized non linear gradient ph 3 10 strips, by Cytiva (1 mentions)
Protein ief cell, by Bio-Rad (1 mentions)
Complete protease, by Merck Group (1 mentions)
Pierce coomassie plus, by Thermo Fisher Scientific (1 mentions)
Phosphatase, by Merck Group (1 mentions)
Western lightning plus ecl reagent, by PerkinElmer (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Quantity one, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
21 protocols using gs710 densitometer
1
Western Blot Analysis of β-Catenin
2
Proteomic Analysis of APL-Treated YD-10B Cells
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YD-10B cells were treated with APL for 72 h. To prepare samples, the cells were washed twice with 1/4 diluted phosphate-buffered saline. Protein extraction and quantification were conducted at the Yonsei Proteome Research Center [20 (link)]. 2D-GE was performed as described, and 540 μg of the protein was loaded on the gel. Aliquots of the sample buffer (7 M urea, 2 M thiourea, 4.5% CHAPS, 100 mM DTE, 40 mM Tris, pH 8.8) were placed on immobilized non-linear gradient pH 3–10 strips (Amersham Biosciences, Uppsala, Sweden). Isoelectric focusing was performed at 80,000 Vh. The second dimension was analyzed on 9–16% linear gradient polyacrylamide gels (18 cm × 20 cm × 1.5 mm) at a constant current of 40 mA for each gel for approximately 5 h. After protein fixation in 40% methanol and 5% phosphoric acid for 1 h, the gels were stained with Coomassie Brilliant Blue G-250 for 12 h. The gels were destained with water and scanned using Bio-Rad GS710 densitometer (Bio-Rad Laboratories, Inc., Richmond, CA, USA), which were converted into electronic files and analyzed using the ImageMaster Platinum 5.0 image analysis program (Amersham Biosciences, Amersham, UK).
3
Two-Dimensional Gel Electrophoresis of Brucella abortus
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The 2-DE was carried out essentially as described. B. abortus wild-type and 5 mutants in sample buffer (7 M urea, 2 M thiourea, 4.5% CHAPS, 100 mM DTE, 40 mM Tris, pH 8.8) were applied to immobilized pH 3 to 10 nonlinear gradient strips (Amersham Biosciences, Sweden) for isoelectric focus (IEF). Similar amounts of each protein from B. abortus wild-type and mutants underwent 2-DE analysis after quantification. IEF was performed at 80,000 Vh. The second dimension was analyzed on 9% to 16% linear gradient polyacrylamide gels (18 cm × 20 cm × 1.5 cm) at constant 40 mA per gel for approximately 5 h. After protein fixation in 40% methanol and 5% phosphoric acid for 1 h, the gels were stained with Coomassie brilliant blue G-250 for 12 h. The gels were then destained with distilled water, scanned in a Bio-Rad GS710 densitometer (Bio-Rad, USA) and converted to electronic files, which were then analyzed by using the Image Master Platinum 5.0 image analysis program (Amersham Biosciences) [16 (link)17 (link)].
4
Comparative Proteomic Analysis of B. henselae
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Whole cell proteins were separated in the first dimension by using immobilized pH 3 to 10 nonlinear gradient strips (Amersham Biosciences, UK). Isoelectric focusing of the protein-containing samples was performed in a protein IEF cell (BioRad). Afterward, seconddimension analysis was performed on 9% to 16% linear gradient polyacrylamide gels, and protein fixation was performed. They were scanned in a Biorad GS710 densitometer (BioRad) and the results converted into electronic files and were analyzed by using the Image Master Platinum 5.0 image analysis program (Amersham Biosciences). Analysis was conducted to identify spots with a minimum 2-fold increased or decreased difference between B. henselae Houston-1 (ATCC49882) and kanamycin-resistant omp43 deficient B. henselae (Δomp43).
5
Quantification of Plasma TK1 Levels in Mice
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Plasma thymidine kinase 1 (Tk1) levels in mice were measured by the enhanced chemiluminescent dot blot assay. Three μl of serum from control and SMA-RL7 treated mice as well as the recombinant human TK1 protein (rHTK1) standard (0.00056–0.18 μM), were applied to a nitrocellulose membrane and allowed to air dry. Membranes were then blocked with 10% non-fat milk in TBS for 1 h and washed 3× with TBS for 5 min. Membranes were incubated with anti-TK1 monoclonal antibody (1:500 in 5% BSA) at room temperature for 2 h. Membranes were then incubated (1 h) with biotinylated anti-mouse secondary antibody (1:1000) at room temperature, washed with TBST (5×) followed by streptavidin-conjugated HRP for 30 min and washed 5× with TBST. Membranes were then developed with SuperSignal substrate. Signal intensity was visualized on radiographic film and quantified with a GS-710 densitometer (Bio-Rad). PlasmaTK1 was determined using linear regression as a function of intensity and concentration of rHTK1. Protein extracts from tumors were prepared as previously described [13 (link),16 (link)]. The density of each band was normalized to a β-tubulin loading control.
6
2-DE Proteomic Analysis Protocol
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All 2-DE was performed as described [14 (link)]. Briefly, aliquots in sample buffer (260 μg) were applied to immobilized pH 3–10 nonlinear gradient strips (Amersham Biosciences, Uppsala, Sweden). Isoelectric focusing was performed at 80,000 Vh. The second dimension was analyzed on 9–16% linear gradient polyacrylamide gels (18 cm × 20 cm × 1.5 cm) at a constant 40 mA per gel for 5 hours. After protein fixation in 40% methanol and 5% phosphoric acid for 1 hour, gels were stained with Coomassie Brilliant Blue G-250 for 12 hours. Gels were then destained with H2O, scanned in a Bio-Rad GS710 densitometer (Bio-Rad, Hercules, CA, USA), and converted into electronic files (12 bit tiff).
7
Western Blot Analysis of Cellular Proteins
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Samples containing 10 μg of protein from cellular extracts were resolved using SDS-PAGE at 100 V for E-cadherin and vimentin, whereas 40 μg of protein were resolved for Erk, pErk, Akt and pAkt. After the sample ran down to the bottom of the gel, the membrane was removed and transferred into the transfer buffer. A sandwich containing equilibrated fibre pad and blotting paper and an activated membrane was made in cassettes. A voltage of 100 V was set, and the transfer process was carried out for 90 min. The membrane was blocked with BSA blocking buffer 1 ×, followed by a primary antibody incubation (pErk, Erk, pAkt, Akt, E-cadherin and vimentin) overnight, after which the membrane was washed six times with TBST and incubated with secondary antibody for a period of one hour. After six further washes with TBS, chemiluminescent solutions were then added to the membrane, and X-ray films were exposed to the membrane, after which the films were developed. The films were analysed using a BioRad GS710 densitometer (Hercules, CA, USA), and the protein density was calculated as a percentage of β-actin or β-tubulin. Three independent experiments were carried out.
8
Western Blotting Analysis of PMP22 in Sciatic Nerve
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For Western blotting, mouse sciatic nerve lysates from wild-type, C22, or PMP22-knockout mice (4 μg/lane) were blotted with anti-human PMP22-specific polyclonal antibody.25 Confluent cultures of dermal fibroblasts, after 48 hours incubation in 2% FCS containing medium, were lyzed in sample buffer [62.5 mmol/L Tris, 3% SDS, and 10% glycerol, with complete protease (Roche) and phosphatase (Sigma-Aldrich, St. Louis, MO) inhibitors]. Protein concentration was determined using the Bradford method (Pierce Coomassie Plus; Thermo Fisher Scientific, Waltham, MA), and samples were denatured by adding β-mercaptoethanol and boiling for 10 minutes. Equal protein amounts were resolved on SDS polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were rinsed in Tris-buffered saline with 0.05% Tween-20, blocked in 5% nonfat milk, and then probed with the indicated primary antibodies (Table 2 ) overnight at 4°C. Bound antibodies were detected using Western Lightning Plus ECL reagents (Perkin Elmer, Waltham, MA). Films were digitally imaged using a GS-710 densitometer (Bio-Rad Laboratories, Hercules, CA) or a ChemiDoc MP Imaging System (Bio-Rad Laboratories) and analyzed using ImageJ software version 1.45 (NIH, Bethesda, MD; http://imagej.nih.gov/ij ). All biochemical experiments were repeated at least three times using protein lysates from independent cultures.
9
Comprehensive Protein Separation by 2D-Gel Electrophoresis
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Two-dimensional gel electrophoresis (2-DE) was conducted as reported. Briefly, aliquots in sample buffer (7 M urea, 2 M thiourea, 4.5% CHAPS, 100 mM DTE, 40 mM Tris, pH 8.8) were added to immobilized pH 3–10 nonlinear gradient strips (Amersham Biosciences, Uppsala, Sweden). Isoelectrofocusing (IEF) was performed at 80,000 Vh. The second dimension was analyzed on 9–16% linear gradient polyacrylamide gels (18 cm × 20 cm × 1.5 mm) at a constant 40 mA per gel for approximately 5 h. After protein fixation in 40% methanol and 5% phosphoric acid for 1 h, the gels were stained with CBB G-250 for 12 h. The gels were destained with H2O, scanned with a GS710 densitometer (Bio-Rad, Richmond, CA, USA) and converted to electronic files, which were then analyzed with an Image Master Platinum 5.0 image analysis program (Amersham Biosciences, Amersham, UK).
10
Flagellin-induced IκBα degradation in MCF7 cells
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MCF7 cells were pre-incubated with or without 10 nM E2 for 24 h before the stimulation with 100 ng/ml of flagellin. Following treatment, cells were harvested at 0, 15, 30, 45, 60 and 90 min in 200 μl lysis buffer (40 mM Tris-Cl, pH 6.8, 1% w/v SDS, 1% v/v glycerol, 1% v/v β-mercaptoethanol, 0.01% w/v bromophenol blue). The lysates were boiled for 10 min and the proteins separated by polyacrylamide gel (12%) electrophoresis and transferred to nitrocellulose membranes. Membranes were probed using the following antibodies: anti-IκBα (#9242, Cell Signalling, MA, USA) and anti-alpha tubulin (#2144, Cell Signalling, MA, USA). Band intensity was quantified using a BioRad GS-710 Densitometer and Quantity One software (Version 4.5.0).
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