At the end of scanning, proteins were fixed with 50% ethanol containing 10% acetic acid, and the gels were stained in a freshly made ammoniacal silver nitrate solution. The silver-stained gel images were scanned using an ImageScanner (GE Healthcare Bio-Sciences).
Imagescanner
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom
The ImageScanner is a versatile laboratory equipment designed for high-quality image capture and analysis. It features a compact and user-friendly design, enabling efficient scanning and digitization of various sample types. The ImageScanner is capable of producing detailed and accurate digital representations of samples, supporting a range of scientific and research applications.
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Lab products found in correlation
Image master 2d platinum, by GE Healthcare (5 mentions)
Immobiline drystrip, by GE Healthcare (2 mentions)
2 d clean up kit, by GE Healthcare (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ipg strip, by GE Healthcare (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Criterion cell, by Bio-Rad (1 mentions)
Bio safe coomassie blue stain, by Bio-Rad (1 mentions)
3 3 diaminobenzidine tetrahydrochloride dab, by Merck Group (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Semi dry transfer cell, by Bio-Rad (1 mentions)
Hrp conjugated goat anti human igg, by Merck Group (1 mentions)
Imagemaster 2d platinum 6, by GE Healthcare (1 mentions)
Mini protean cell, by Bio-Rad (1 mentions)
Ipg strip, by Bio-Rad (1 mentions)
Imagemaster 2d platinum v 6, by GE Healthcare (1 mentions)
Brilliant blue g colloidal concentrate, by Merck Group (1 mentions)
Quantity one software, by GE Healthcare (1 mentions)
Tris mops sds running buffer, by GenScript (1 mentions)
Coomassie brilliant blue, by Merck Group (1 mentions)
54 protocols using imagescanner
1
Silver Staining of Protein Gels
2
Proteomics Analysis of Rubisco Depletion
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One-dimensional gel electrophoresis was used to resolve proteins from the bound and flow-through fractions obtained during Rubisco depletion on the Seppro column. Ten μl of each fraction containing approximately 10 μg of protein was mixed with 10 μl of gel sample buffer (0.2 M Tris-HCl, pH 6.8, 2% SDS, 10% glycerol,0.02% bromophenol blue) and separated on a 1.0 mm, 12.5% Criterion Tris/HCl gel in a Criterion Cell (Bio-Rad) (13.3 cm × 8.7 cm) at a constant voltage of 150 V. The separated proteins were visualized using Bio-Safe Coomassie Blue stain (Bio-Rad).
For phosphoproteome analysis, 200 μl (200 μg) of IMAC-enriched phosphoprotein sample was mixed with 200 μl of rehydration buffer (7 M urea, 2 M thiorea, 2% CHAPS, 10 mM DTT, 0.5% IPG buffer, pH 3–10), resolved by 2-DE and visualized by silver staining 10. Gel images were recorded using an ImageScanner (GE Healthcare) and Phoretix 2D software (v2004) was used to measure the total number of protein spots visualized in each 2-DE gel image. Proteins of interest were excised manually from each gel and digested with trypsin using a MassPREP protein digestion station, according to the protocol (digestion 5.0) recommended by the manufacturer (Micromass, Manchester, UK). Preparation of tryptic peptide samples for LC-MS/MS analysis was carried out as previously described.[11 (link)]
For phosphoproteome analysis, 200 μl (200 μg) of IMAC-enriched phosphoprotein sample was mixed with 200 μl of rehydration buffer (7 M urea, 2 M thiorea, 2% CHAPS, 10 mM DTT, 0.5% IPG buffer, pH 3–10), resolved by 2-DE and visualized by silver staining 10. Gel images were recorded using an ImageScanner (GE Healthcare) and Phoretix 2D software (v2004) was used to measure the total number of protein spots visualized in each 2-DE gel image. Proteins of interest were excised manually from each gel and digested with trypsin using a MassPREP protein digestion station, according to the protocol (digestion 5.0) recommended by the manufacturer (Micromass, Manchester, UK). Preparation of tryptic peptide samples for LC-MS/MS analysis was carried out as previously described.[11 (link)]
3
SDS-PAGE and Western Blot for Trichinellosis Antigens
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The AW ES antigens (20 μg) were subjected to sodium dodecyl sulfat-polyacrylamide gel (SDS-PAGE) in 5% stacking gels and 12% resolving gels at 80 V for 40 min and 120 V for 90 min. After electrophoresis, one gel was stained in Coomassie brilliant blue R-250 staining solution (Sigma, United States) for 4 h, and the other gel was used for the electrotransfer of proteins on nitrocellulose (NC) membranes (Millipore, United States) at 18 V for 35 min via a semi-dry transfer cell (Bio-Rad, United States) (Wang B. et al., 2013 (link)). Subsequently, the membranes were cut into strips, blocked with 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.6) at 37°C for 1 h, and incubated at 4°C overnight with sera from different patients with trichinellosis at a dilution of 1:100. After being washed in TBST, the strips were incubated with HRP-conjugated goat anti-human IgG (1:5,000 dilutions; Sigma, United States) at 37°C for 1 h. Finally, the immunoreaction was detected with 3, 3-diaminobenzidine tetrahydrochloride (DAB; Sigma, United States). The gel and membranes were scanned using ImageScanner (GE healthcare, United States) and the MW of these bands was analyzed by AlphaView software (ProteinSimple, Santa Clara, CA, United States).
4
Gel-Based Protein Quantification and Comparison
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Stained gels were scanned using an image scanner (GE Healthcare, Bio-Sciences, Uppsala, Sweden). Images were analyzed with ImageMaster and Melanie analysis software (Amersham Biosciences, Piscataway, NJ, USA, 2011), including spot detection, background subtraction, volumetric quantification, and matching. Protein spots were selected based on a fold change of ≥ 2 or ≤ 0.5. A threshold of p ≤ 0.05 was used to select differentially expressed protein spots.
5
Proteomic Analysis of Excretory-Secretory Proteins
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The ES proteins were precipitated using trichloroacetic acid (TCA) and acetone using a previously described method with some modifications [27 (link)]. The electrophoresis was performed as described previously [17 (link),28 (link)]. Briefly, 300 or 200 μg of ES proteins were loaded onto 11-cm pH 4–7 immobilized pH gradient (IPG) strips (Bio-Rad, USA) and separated by isoelectric focusing (IEF). IEF was performed using a Protean IEF Cell at 20°C as follows: S1: 50 V, 12 h; S2:250 V, 30 min; S3: 1 000 V, 30 min; S4: 8 000 V, 4 h; and S5: 8 000 V, 40 000 Vh (using a limit of 50 μA/strip). SDS-PAGE was performed with 10% gels using a Mini Protean cell (Bio-Rad, USA). Three replicates were run for the sample. After 2D gel electrophoresis, proteins were either stained with Coomassie blue R-250 for proteomic analysis or used for immunoblotting as previously described [29 (link)]. Both the 2-DE and immunoblotting tests were repeated three times, with no variation in results observed. Images of immunoblots were captured using ImageScanner (GE healthcare, USA) and aligned with equivalent protein stained 2-DE gels using Image Master 2D Platinum 6.0 (GE healthcare, USA).
6
Proteomic Analysis of Antimicrobial Responses
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Cells in the exponential growth phase were harvested from BHI broth, washed in Tris-HCl 50 mM (pH 7.5) and used to generate proteins extracts as described by De Angelis et al. (2001) (link). Equivalent amounts of total protein (60 μg for analytical runs or 200 μg for preparative runs for protein identification) were used for each electrophoretic run. The 2-DE was performed essentially as described by Görg et al. (1988) (link) and Hochstrasser et al. (1988) (link) using a Pharmacia 2-D-Electro Focusing (EF) system (GE Healthcare, Milano, Italy). Gels were stained using Brilliant Blue G-Colloidal Concentrate (Sigma) or an MS-compatible silver method. The protein maps were scanned using LabScan on an ImageScanner (GE Healthcare) and were analyzed using ImageMaster 2D Platinum v.6.0 (GE Healthcare). Three gels from three independent experiments were analyzed, and the spot intensities were normalized (De Angelis et al., 2001 (link)), with the spot quantification for each gel calculated as a relative volume (% vol) that corresponded to the volume of each spot divided by the total volume over the entire image. The comparison between different conditions for the amount of the same protein was carried out as the rate of the relative volume of the same spot found in control (untreated cells) and ethanol or other antimicrobials treated cells (Siragusa et al., 2014 (link)).
7
SDS-PAGE Protein Analysis Protocol
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the procedure of Xiong et al. with minor modifications.16 (link) Prior to electrophoresis, a given volume of protein sample was mixed with the SDS-PAGE sample buffer (4% SDS, 20% glycerol, and 0.125 M Tris, pH 6.8), with or without 5% βME, and made up to a final protein concentration of 1.5 mg mL−1. For samples without βME, 0.5 mM N-ethylmaleimide (a thiol blocking agent) was added to prevent disulfide artifacts. All mixtures were heated in boiling water (100 °C) for 5 min. A 12 μL sample aliquot (18 μg of protein) was loaded onto the 4–12% precast gel. The gels were run in Tris-MOPS-SDS running buffer (1 L; GenScript, USA) at 140 V for 1.5 h. Proteins were then stained with Coomassie Brilliant Blue R250 for 0.5 h and destained until the bands were clear. Gel images were captured using an image scanner (GE Healthcare, Little Chalfont, UK), and the band intensities were quantified using Quantity One software (GE Healthcare, Little Chalfont, UK). The relative intensity of each band was calculated from the actual intensity of the measured band relative to the actual intensity of the 130 kDa band in the prestained calibration marker.
8
SDS-PAGE Protein Separation Protocol
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Reconstituted depleted serum aliquots containing 10 µg of total protein were separated by SDS-PAGE on 12% polyacrylamide gels. The gels were post-stained with Coomassie Brilliant Blue (Sigma-Aldrich, St Louis, MO, USA) and the stained gels were documented using an ImageScanner (GE Healthcare, Chicago, IL, USA).
9
Western Blot Analysis of MKL1 and GAPDH
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Protein extracts (10 μg) prepared with RIPA lysis buffer were resolved on a 12% SDS-PAGE gel, and transferred to an Immobilon-P PVDF transfer membrane (Millipore, Bedford, MA) by electro-blotting. After blocking with 5% non-fat milk, membranes were incubated overnight with a 1:1000 dilution of antibodies at 4 °C. Blots were then incubated with peroxidase-conjugated anti-mouse or anti-rabbit IgG (KPL, Gaithersburg, MD) for 1 h at RT at a 1:1000 dilution and then developed using a Super Signal West Pico kit (Pierce Biotechnology). Immunoblots were scanned using an Image Scanner (GE healthcare). Blot densitometry analysis was performed using Image J (National Institutes of Health).The following antibodies were used for Western blot analyses: rabbit anti-MKL1 polyclonal (Abcam), mouse anti-GAPDH polyclonal (Sigma).All the analysis was performed in triplicate.
10
Gel-based Proteomic Workflow Analysis
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All 60 stained gels (15 populations × 4 biological replicates) were scanned with ImageScanner (GE Healthcare) at 300 dpi and the gel images were subsequently analyzed using Progenesis SameSpots software (Nonlinear Dynamics, Newcastle, UK). A single aberrant gel image was deemed oversaturated and was excluded from further analysis. All gel images were aligned against a reference gel, using a semi-automated procedure in which 30 landmarks were manually added to guide the automated alignment. Protein spot detection on the gel images was performed using default setting for detection, background subtraction, normalization and matching. Spots detected in all, or all but one, gels from a given population were considered valid protein spots. Each protein spot was carefully inspected in detail, based on peak height and 3D visualization, and then manually edited to exclude artifacts if needed (i.e., splitting of closely located but distinct spots or merging of single true spots). Normalization was restored following editing. The normalized volume of each protein spot from each of the gel images was used as a measure of relative protein abundance in subsequent statistical analyses.
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