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Molecular imager versadoc mp 5000 system

Manufactured by Bio-Rad
Sourced in United States

The Molecular Imager VersaDoc MP 5000 System is a gel documentation and molecular imaging system designed for a variety of applications in life science research. The system features a high-resolution camera and advanced optics to capture images of stained gels, blots, and other samples. It supports various detection methods, including chemiluminescence, fluorescence, and colorimetric.

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21 protocols using molecular imager versadoc mp 5000 system

1

Protein Separation by SDS-PAGE

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The electrophoretic separation was performed according to the protocol of Sanchón et al. 30 Samples and the marker (Precision Plus Protein™ Unstained protein standards, Biorad, Hercules, CA, USA) were loaded on 12% bis-tris Criterion™ XT gel (Biorad). Gels where then stained with Coomassie Blu (Instant ble, Expedeon, Swavesey, UK). Images were acquired with a Molecular Imager VersaDocTM MP 5000 system (Bio-Rad) and using the software QuantityOne® 1-D (Bio-Rad).
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2

SDS-PAGE Protein Separation and Analysis

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SDS-PAGE was performed as previously described 16 but protein digests were dissolved at 0.7 mg of protein/mL in sample buffer. Gels were stained with Coomasie Blue (Instant Blue, Expedeon, Swavesey, UK) and images were taken with a Molecular Imager VersaDoc TM MP 5000 system (Bio-Rad, Hercules, CA, USA) and processed with Quantity One 1-D analysis software (Bio-Rad).
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3

SDS-PAGE Protein Separation and Visualization

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The electrophoresis assay was performed on Precast Criterion XT 4–12% Bis-Tris gels using the Criterion cell (Bio-Rad, Hercules, CA, USA) as reported by Sanchón et al. [16 (link)]. The samples were dissolved at 0.8 mg of protein/mL in a buffer containing 0.05 M Tris-HCl, pH 6.8, 8% (v/v) glycerol, 1.6% (w/v) SDS, 2% (v/v) β-mercaptoethanol, and 0.002% (w/v) bromophenol blue. The concentration was calculated at 0.8 mg protein/mL buffer solution. Then, they were heated at 95 °C for 4 min, and 45 μL was loaded onto the 4–12% Bis-Tris polyacrylamide gel as well as 15 μL of the molecular weight marker (Precision Plus Protein unstained Standard (Bio-Rad, Bio-Rad, Hercules CA, USA). Electrophoretic separation was carried out using XT-MES as a running buffer (BioRad). After 5 min at 100, 150 V were applied. The gels were washed with Milli-Q water and were stained with Coomassie blue G-250. Images were taken with a Molecular Imager® VersaDoc™ MP 5000 system (Bio-Rad, Hercules, CA, USA) and processed with Quantity One®1-D analysis software (Bio-Rad Laboratories S.A, Madrid, Spain).
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4

Quantifying Adipose Tissue Cytokines

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To measure IL-1β, IL-6, IL-8, and IL-15 concentrations in serum and in protein extracts from adipose tissues, an ELISA-based chemiluminescent custom-made Q-plex Custom array (Quansys Bioscience, West Logan, UT, USA) was used. Luminescence was assessed in the Molecular Imager Versa Doc™ MP 5000 System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s guidelines. Results were analyzed with the Q-View software version 2.17 (Quansys Bioscience, West Logan, UT, USA). Measurements of interleukin concentrations in adipose tissues were normalized to total protein concentration in protein extracts obtained from these tissues. The mean protein concentrations in extracts from visceral (VAT) and subcutaneous (SAT) tissues obtained from obese (O) and normal-weight (N) individuals were similar (p > 0.05, Supplementary Figure S1).
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5

Protein Expression Analysis in Renal Tissues

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Renal tissues were washed by PBS and lysed in lysis buffer (Beyotime, Shanghai, China). The lysates were then incubated on ice for 30 min and oscillated for 30 s. The cell lysates were centrifuged at 10,000 g for 30 min at 4°C. The supernatant was collected to measure the protein concentrations by BCA kit (Solarbio, Beijing, China). The proteins separated by the SDS-PAGE were transferred onto polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK). After being blocked for 1 h, the membranes were incubated overnight at 4°C with primary antibodies as follows: Bcl-2 (1:500, Abcam, Cambridge, UK), Bax (1:500, Abcam), caspase-3 (1:500, Abcam), HIF-1α (1:1000, Abcam), VEGF (1:1000, Abcam), survivin (1:1000, Cell Signaling Technology), p21 (1:1000, Cell Signaling Technology), cyclin D1 (1:1000, Cell Signaling Technology) and GADPH (1:1000, Cell Signaling Technology). Thereafter, the membranes were probed with secondary antibodies (1:1000, Beyotime). The bands were determined by a Molecular Imager VersaDoc MP 5000 System (Bio-Rad, Hercules, CA). The densitometry was determined with a Quantity One (Bio-Rad).
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6

Western Blot Analysis of Liver Samples

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Liver samples were lysed and homogenized in NP40 lysis buffer. After centrifugation (12,000 rpm, 15 min, 4 °C) of the homogenized samples, protein concentration of the supernatant was determined using BIO-RAD DC protein assay kit (BIO-RAD, Hercules, CA). Equal amounts of protein were fractionated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were incubated in Tris-buffered saline (TBS) containing 5 % non-fat milk (for 1 h at room temperature) and then in primary antibodies overnight at 4 °C: HO-1 (1:1,000), SIRT1 (1:1,000) and Beta actin (1:5,000). The following day, the membranes were washed in TBS-Tween and incubated for 1 h at room temperature with the corresponding secondary antibody: goat anti-mouse stabilized peroxidase conjugate (1:1,000) or anti-mouse IgG (whole molecule)-peroxidase (1:80,000). Bands were detected using Molecular Imager VersaDoc™ MP 5000 System and the protein density was measured using the Quantity One 1-D Analysis Software (Bio-Rad, Prague, Czech Republic).
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7

Western Blot Analysis of Clusterin in Prostate Tissues

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Prostate tissues and cells were washed by PBS and lysed in lysis buffer (Beyotime, Beijing, China). Then, the lysates were incubated on ice for 30 min and oscillated for 30 s. After centrifugation at 10 000 g for 30 min at 4°C, the supernatant was collected to measure the protein concentrations by BCA kit (Solarbio, Beijing, China). The proteins separated by the SDS-PAGE were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Then, the membranes were incubated overnight at 4°C for 1 h after blocking with primary anti-clusterin (1: 1000, Santa Cruz Biotechnology) and anti-β-actin (1: 1000, Santa Cruz Biotechnology). The membranes were then probed with anti-rabbit IgG (1: 50 000, Cell Signaling Technology, Beverly, MA, USA). The bands were determined by a Molecular Imager VersaDoc MP 5000 System (Bio-Rad, Hercules, CA) and densitometry was determined with Quantity One (Bio-Rad).
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8

Protein Extraction and Western Blot Analysis

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Renal tissues were washed by PBS and lysed in lysis buffer (Beyotime, Shanghai, China). Then the lysates were incubated on ice for 30 min and oscillated for 30 s. After centrifugation at 10,000 g for 30 min at 4 °C, the supernatant was collected to measure the protein concentrations by BCA kit (Solarbio, Beijing, China). The proteins separated by the SDS-PAGE were transferred onto polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK). Then the membranes were incubated overnight at 4 °C 1 h after blocking with primary antibodies as follows: KNG1 (1:500, Abnova, Jhongli, Taiwan), caspase-3 (1:500, Abcam), caspase-9 (1:500, Abcam), CyclinD1 (1:1000, Abcam), ki67 (1:1000, Abcam), X-linked inhibitor of apoptosis (XIAP) (1:1000, Cell Signaling Technology, Beverly, MA), VEGF (1:1000, Cell Signaling Technology), PI3K (1:1000, Cell Signaling Technology), P-PI3K (1:1000, Cell Signaling Technology), Akt (1:1000, Cell Signaling Technology), p-Akt (1:1000, Cell Signaling Technology) and GAPDH (1:10000, Cell Signaling Technology). Then the membranes were probed with the anti-rabbit IgG (1:50000, Cell Signaling Technology). The bands were determined by a Molecular Imager VersaDoc MP 5000 System (Bio-Rad, Hercules, CA). The densitometry was determined with a Quantity One (Bio-Rad).
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9

SDS-PAGE Protein Separation Protocol

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Samples were dissolved (0.5 mg of protein/mL) in sample buffer and heated at 95°C for 5 min. 25 μL of sample (12.5 μg protein) was loaded on 12% Bis-Tris polyacrilamide gels (Criterion_XT, Bio-Rad, Hercules, CA, USA). Electrophoretic separations were run at 100 V for 5 min and then, at 150 V for 50 min, using the XT MES running buffer (Bio-Rad) in the Criterion Cell (Bio-Rad). The molecular weight Precision Plus Protein™ Unstained standard marker (Bio-Rad) was used. Coomasie Blue (Instant blue, Expedeon, Swavesey, UK) was used to stain the gels, and images were taken with a Molecular Imager ® VersaDoc™ MP 5000 system (Bio-Rad) and processed with Quantity One ® 1-D analysis software (Bio-Rad).
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10

Comprehensive Protein Expression Analysis

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Total protein was extracted in ice-cold radioimmunoprecipitation assay buffer containing protease inhibitors (Beyotime Institute of Biotechnology), and quantified using a BCA protein assay kit. Proteins were denatured, separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane by the wet transfer method. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST) for 2 h at room temperature and incubated with blocking solution containing primary antibody (1:400, anti-collagen III; 1:400, anti-MMP8; 1:400, anti-MMP14; 1:400, anti-TIMP2; 1:400, anti-CSE; 1:400, anti-TGF-β; 1:400, anti-TNF-α; 1:400, anti-NF-κB; 1:400, anti-STAT1/3/5/6; 1:400, anti-JAK-1/2; 1:1,000, anti-eIF2α; 1:1,000, anti-GRP94; 1:1,000, anti-caspase-3; and 1:1,000, anti-Bcl-2) overnight at 4°C. After washing three times with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000) for 1 h at room temperature. Next, the membranes were washed in TBST buffer three times and subjected to chemiluminescence detection assay. The bands were analyzed with a Molecular Imager VersaDoc MP 5000 system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
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