Sample preparation and 2DE gels were carried out according to Bustos et al. (2015 (link)). Isoelectrofocusing (IEF) was performed in IPGphor (GE Healthcare, Uppsala, Sweden) at 53,500 Vh, using the immobilized pH gradient (IPG) strips (Immobiline DryStrip Gels, linear pH 4–7, 18 cm, GE Healthcare; Uppsala, Sweden). For the second dimension, IEF strips were equilibrated at room temperature in 6 M urea, 2% (w/v) SDS, 30% (w/v) glycerol, 50 mMTris-HCl, pH 8.0, containing alternatively 50 mM DTT (15 min) and then 400 mM iodoacetamide (15 min in the dark). Second dimension was performed on homogeneous 12.5% (w/v) polyacrylamide gels at the constant current of 15 mA/gel at 15°C (~16 h) using an Ettan DALTsix Large Vertical System (GE Healthcare, Uppsala, Sweden). Gels were stained with colloidal Coomassie blue Stain according to Candiano et al. (2004 (link)), distained with distilled water. The 2DE maps were digitalized using Image Scanner III LabScan 6.0 (GE Healthcare, Uppsala, Sweden).
Ettan daltsix large vertical system
Manufactured by GE Healthcare
Sourced in Sweden
The Ettan DALTsix Large Vertical System is a laboratory equipment designed for performing large-format two-dimensional (2D) gel electrophoresis. It is capable of simultaneously running up to six large-format polyacrylamide gels for the separation and analysis of complex protein samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagescanner 3 labscan 6, by GE Healthcare (2 mentions)
Immobiline drystrip, by GE Healthcare (2 mentions)
Bio lyte 3 10 ampholyte, by Bio-Rad (2 mentions)
Ettan ipgphor, by GE Healthcare (2 mentions)
Ipgbox, by GE Healthcare (2 mentions)
Ipgphor, by GE Healthcare (1 mentions)
Typhoon trio imager, by GE Healthcare (1 mentions)
Ursolic acid, by Merck Group (1 mentions)
Image master 2d platinum, by GE Healthcare (1 mentions)
Imagescanner, by GE Healthcare (1 mentions)
Ettan ipgphor 2 isoelectric focusing system, by GE Healthcare (1 mentions)
Ettan ipgphor 3, by GE Healthcare (1 mentions)
6 protocols using ettan daltsix large vertical system
1
Two-Dimensional Electrophoresis of Proteins
2
Proteomic Analysis of Protein Changes
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Six replicates from each group were analyzed by DIGE. Lysates containing 800 µg of protein were purified by methanol/chloroform precipitation according to the previous protocol [28 (link)]. Pellets were dissolved in the lysis buffer. Protein concentrations were measured by the Bradford assay and adjusted to 4.5 mg/ml. Labeling of the proteins and separation by 2DE was executed as described in our earlier work [29 (link)]. Briefly, 33 μg of protein from each sample was labeled with 110 pmol of Cy3 or Cy5 and the IS was labeled with Cy2. Samples were combined according to Table S1 in Supplementary file 1 and loaded onto 24 cm nonlinear pH 3–10 Immobiline DryStrips (GE Healthcare). IEF was performed on a PROTEAN® i12™ IEF System for a total of 78 000 Vhr. SDS-PAGE was carried out using an Ettan DALTsix Large Vertical System (GE Healthcare). Fluorescent images of the DIGE gels were obtained using the Typhoon Trio + Imager (GE Healthcare) at a resolution of 100 dpc. Next, the gels were stained with a lab-made ruthenium II tris-(bathophenanthroline disulfonate) and scanned again [30 (link)].
3
Proteomic Analysis of Ursolic Acid Response
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The bacteria were treated with ursolic acid (Sigma, St. Louis, MO, USA) at a concentration corresponding to 4 × MIC for 60 min according to previous reports [9 (link),16 (link)]. This concentration decreases the growth rate of a mid-log-phase culture by 50% (Table 3 ) and could therefore be expected to trigger major changes in protein expression. Two-dimensional gel electrophoresis was performed by using the Ettan™ IPGphor II Isoelectric Focusing system (GE Healthcare, Piscataway, NJ, USA) according to the instructions of the manufacturer. Protein samples (300 μg) were separated by using IPG strips in pH range of 3–10. Isoelectric focusing was performed by using 7 M urea, 2 M thiourea, 4% CHAPS and 40 mM DTT with increasing voltage. Rod gels were soaked for 15 min at ambient temperature in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 10 mg/mL DTT) and applied to a second dimension using a commercially available 12% Tris-Glycine SDS gel (13 cm × 13 cm × 1.0 mm) (Pharmacia, Uppsala, Sweden) with an Ettan™ DALTsix Large Vertical System (GE Healthcare, Uppsala, Sweden). Image was scanned by ImageScanner™ (GE Healthcare, Uppsala, Sweden) and analyzed by ImageMaster™ 2D Platinum (GE Healthcare, Uppsala, Sweden).
4
Two-Dimensional Protein Separation Protocol
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For each strip, 150 μg of protein (50 μg from each dye) were loaded along with rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.001% bromophenol blue, 0.5% Bio-lyte 3/10 ampholyte (Bio-Rad Laboratories, Hercules, California, USA) to complete 450 μl. Passive rehydration was conducted for 15 h on 24 cm Immobiline™ Drystrips (GE Healthcare, Little Chalfont, UK) with linear pH 4–7, on an IPG Box (GE Healthcare, Little Chalfont, UK). Following, isoelectric focusing (IEF) was performed in 5 steps: 500 V gradient 1 h, 500 V step-n-hold 1 h, 1000 V gradient 1 h, 8000 V gradient 3 h and 8000 V step-n-hold 5 h40 for a total of 60.000 Vhr using Ettan IPGphor at 20 °C (GE Healthcare, Little Chalfont, UK). Focused strips were reduced and alkylated with 6 ml of equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol and 2% SDS) with 1% (w/v) dithiothreitol (DTT) or 2.5% (w/v) iodoacetamide (IAA) respectively for 15 min each, in constant agitation. Strips were then loaded onto 12.5% Tris-HCl SDS-PAGE gels and ran in an Ettan DALTsix Large Vertical System (GE Healthcare, Little Chalfont, UK) at 10 mA/gel for 1 h followed by 60 mA/gel until the bromophenol blue line reaches the end of the gel, using a standard Tris-Glycine-SDS running buffer.
5
2D Gel Electrophoresis of Proteins
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Sample preparation and 2DE gels were carried out as previously described Bustos et al.(2015) . Briefly, 600 μg of proteins were placed in each immobilized pH gradient (IPG) strip (Immobiline DryStrip Gels, linear pH 4-7, 18 cm, GE Healthcare; Uppsala, Sweden) and Isoelectrofocusing (IEF) was performed in Ettan IPGphor 3 (GE Healthcare, Uppsala, Sweden) at 53,500 Vh. For the second dimension, IPG strips were equilibrated at room temperature in 6 M urea, 2 % SDS, 30 % glycerol, 50 mM Tris-HCl, pH 8.0, containing alternatively 50 mM DTT (15 min) and then 400 mM iodoacetamide (15 min in the dark). Second dimension was performed on homogeneous 12.5 % polyacrylamide gels at the constant current of 15 mA/gel at 15 ºC (approximately 16 h) using an Ettan DALTsix Large Vertical System (GE Healthcare, Uppsala, Sweden).
Gels were stained with colloidal Coomassie blue Stain according to Candiano et al. (2004) , distained with distilled water. The 2DE maps were digitalized using Image Scanner III LabScan 6.0 (GE Healthcare, Uppsala, Sweden).
Gels were stained with colloidal Coomassie blue Stain according to Candiano et al. (2004) , distained with distilled water. The 2DE maps were digitalized using Image Scanner III LabScan 6.0 (GE Healthcare, Uppsala, Sweden).
6
2D-DIGE Protein Separation Protocol
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Protein separation was achieved by the two-dimensional difference gel electrophoresis (2D-DIGE) method, as follows. DIGE minimal labeling was performed as described in (Raposo de Magalhães et al., 2020b) . Passive rehydration was carried out for 18 h on 24 cm Immobiline TM Drystrips (GE Healthcare) with linear pH 4-7, on an IPG Box (GE Healthcare). A total of 150 µg of protein (50 µg from each dye) were loaded onto each strip, along with a rehydration buffer [8 M urea, 2% CHAPS detergent, 50 mM dithiothreitol (DTT), 0.001% bromophenol blue, 0.5% Bio-lyte 3/10 ampholyte (Bio-Rad)] to fulfill 450 µl. Isoelectric focusing (IEF) was performed in 4 steps: 250 V gradient 4 h, 1000 V gradient 6 h, 8000 V gradient 3 h 40 min, 8000 V step-n-hold 3 h 20 min for a total of 50,000 Vhr using the Ettan IPGphor at 20 • C (GE Healthcare). Focused strips were reduced and alkylated with 6 ml of equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% (v/v) glycerol and 2% SDS) with 1% (w/v) DTT or 2.5% (w/v) iodoacetamide (IAA) respectively for 15 min each, in constant agitation. Strips were then loaded onto 12.5% Tris-HCl SDS-PAGE in-house gels and ran in an Ettan DALTsix Large Vertical System (GE Healthcare), using a standard Tris-Glycine-SDS running buffer, at 10 mA/gel for 1 h followed by 60 mA/gel until the bromophenol blue line reaches the end of the gel.
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