Labelled samples in DIGE lysis buffer were reduced with 10 mg/mL dithiothreitol (DTT) then applied via cup loading to 24 cm pH 3-10NL IPG strips. Iso-electric focusing (IEF) was performed on an IPGPhor3 instrument (GE Healthcare) with the following run conditions: 1) Step and Hold – 150 V for 3 h, 2) Step and Hold – 300 V for 3 h, 3) Gradient – 1000 V 6 h, 4) Gradient – 10,000 V 1 h, 5) Step and Hold 10,000 V 5 h. After focusing for a total of 50,000 Vh, strips were incubated in equilibration buffer (6 M urea, 2 % SDS, 50 mM Tris–HCl pH 8.8, 0.02 % bromophenol blue, 30 % glycerol) with 1 % DTT for 15 min, and then another 15 min in equilibration buffer containing 2.5 % iodoacetamide. Strips were then sealed with 0.5 % agarose on top of 12.5 % SDS-PAGE gels lab cast in low fluorescence glass plates, and run using the Ettan Dalt II system (GE Healthcare) under the following conditions: Step 1) 1 W/gel for 1 h, Step 2) 17 W/gel for 5 h at 25 °C.
Ettan dalt 2 system
Manufactured by GE Healthcare
The Ettan DALT II system is a two-dimensional gel electrophoresis (2D-PAGE) system designed for the separation and analysis of complex protein samples. It enables the separation of proteins based on their isoelectric point and molecular weight, providing a comprehensive view of the proteome.
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Lab products found in correlation
Imagemaster 2d platinum 6, by GE Healthcare (2 mentions)
Ipgphor ief system, by GE Healthcare (1 mentions)
Imagemaster 2d elite software, by GE Healthcare (1 mentions)
Ettan ipgphor 3 isoelectric focusing unit, by GE Healthcare (1 mentions)
Ipg strip, by GE Healthcare (1 mentions)
Ettan ipgphor 3 ief system, by GE Healthcare (1 mentions)
Protein ief cell, by GE Healthcare (1 mentions)
Ipg drystrip, by GE Healthcare (1 mentions)
Ipgphor system, by GE Healthcare (1 mentions)
Coomassie blue g 250, by Bio-Rad (1 mentions)
7 protocols using ettan dalt 2 system
1
2D-DIGE Protein Separation Protocol
2
Quantitative 2-DE Proteome Analysis
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2-DE samples were prepared according to the previously described methods.25 (link), 26 , 27 (link), 28 (link), 29 (link) After delipidation and desalting, the protein concentration of the samples was measured via a modified Bradford method using BSA as a standard.30 (link) Immobilized DryStrips (24 cm, pH 3-10) utilized for isoelectric focusing (IEF) were rehydrated with 40 μg of protein in 450 μl of solubilization solution containing 8 M of urea, 2% of CHAPS, 1% of immobilized pH gradient (IPG) buffer (pH 3-10), 13 mM of dithiothreitol (DTT) and a trace of bromophenol blue for 5 h without current and for another 7 h at 80 V. IEF was conducted using the IPGphor IEF system (GE Healthcare, Uppsala, Sweden) for 120 000 Vhr. The second dimension was run on 12.5% of SDS-PAGE with an Ettan DALT II system (GE Healthcare). Proteins were visualized via silver staining. All experiments were conducted in triplicate. Computer analyses of the 2-DE images were conducted using an ImageMaster 2D Elite Software (GE Healthcare). The expression levels of the spots were determined in accordance with the relative spot volume of each protein, as compared with the normalized volumes of proteins.31 (link)
3
Proteomic Analysis of Soluble and Insoluble Proteins
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Each prepared soluble and insoluble protein was separated by 2-DE individually and the differentially expressed proteins were identified by MALDI-TOF MS using a method described previously (21 (link)). In brief, samples of 1.3 mg soluble or insoluble protein were loaded on an immobilized pH gradient gel strip (pH 3–10 NL, 24 cm; GE Healthcare) using the in-gel rehydration mode. Proteins were separated by first dimensional separation (Ettan IPGphor 3 Isoelectric Focusing Unit; GE Healthcare), and 13% total monomer concentration and 3% weight percentage of crosslinker second dimensional separation (Ettan DALT II system; GE Healthcare). The separated protein spots were visualized using colloidal Coomassie blue staining according to the manufacturer's protocol (GE Healthcare), and the resultant images were analyzed using ImageMaster 2D Platinum 6.0 software (GE Healthcare), according to manufacturer's protocol. Statistical analysis was carried out using the t-test and false discovery rate as described by Biron et al (22 (link)). The differentially expressed protein spots were cut manually from the gels with a stainless-steel scalpel. The in-gel digestion and MALDI-TOF MS of each excised protein spot were performed by the National Center of Biomedical Analysis (Beijing, China).
4
Multidimensional Protein Separation and Visualization
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In the first dimension, total whole-cell protein (250 μg) was loaded onto the IPG strips (24 cm, pH 4–7, GE Healthcare) which had been rehydrated 14 h with 120 mL rehydration solution (7 mol/L urea, 2 mol/L thiourea, 18 mmol/L DTT, 2% Bio-Lyte, 2% (w/v) CHAPS, and 0.002% (w/v) bromophenol blue). Isoelectric focusing was performed on an EttanIPGphor 3 IEF system (GE Healthcare) for a total of 80 kVh at 20 °C. The voltage was set at 50 V for 10 h, 250 V for 3 h, 500 V for 3 h, 1000 V for 1 h, and 8000 V for 1 h, followed by 8000 V until final volt-hours were reached. Subsequently, the strips were equilibrated for 15 min in 2% (w/v) DTT in equilibration buffer (6 mol/L urea, 75 mmol/L Tris–HCl (pH 8.8), 30% (v/v) glycerol and 2% (w/v) SDS) followed by 15 min in 2.5% (w/v) IAA in equilibration buffer. The strips were then transferred to 12.5% (w/v) SDS-polyacrylamide gels. The second dimension electrophoresis was carried out in an Ettan DALTII system (GE Healthcare) with a constant power of 5 W per gel for the first 30 min, followed by 12 W per gel for 6.5–7.5 h until the bromophenol blue front reached the bottom of the gels. The gels were placed into fixative solutions (10% acetic acid,40% methanol) overnight and then stained with 0.25% (w/v) silver nitrate.14 The biological replicates were performed for each treatment at least three times.
5
2D Gel Electrophoresis Protocol
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After IEF, an Ettan DALT II system (GE Healthcare, up to 12 gels at a time) was used. An Ettan DALTsix multiple casters (GE Healthcare) was used to cast the 12% PAGE resolving gel (250 × 215 × 1.0 mm). The resolving-gel solution was made by mixing 75 ml of 1.5 mol/L Tris-HCl pH 8.8, 90 ml of 400 g/L acrylamide/bis-acrylamide (29:1 = weight:weight, cross-linking ratio = 3.3%), 3 ml of 10% ammonium persulfate, 150 ml deionized distilled water (ddH2O), and 50 μl of tetramethylethylenediamine. The IPG strip was equilibrated in 15 ml of reducing equilibration buffer (15 min) that consisted of a trace of bromphenol blue, 375 mmol/L Tris-HCl pH 8.8, 2% w/v SDS, and 20% v/v glycerol. The IPG strip was equilibrated in 15 ml of alkylation equilibrium solution (15 min) that consisted of 2.5% w/v iodoacetamide instead of 2% w/v DTT. A boiled solution that consisted of 1% w/v agarose solution was used to seal the equilibrated IPG strip onto the top of the resolving gel. 2DGE was performed in 25 L of Tris-glycine-SDS electrophoresis buffer that consisted of 192 mmol/L glycine, 25 mmol/L Tris-base, and 0.1 % w/v SDS at 25°C with a constant voltage (250 V, 360 min).
6
Two-Dimensional Gel Electrophoresis Workflow
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The 2-DE (two-dimensional gel electrophoresis) was performed using the immobiline/polyacrylamide system. The isoelectric focusing (IEF) was performed using IPG DryStrips (13 cm; IPGphor; GE Healthcare). Protein samples (200 μg) were applied to the IPG strips using the in-gel sample rehydration technique according to the manufacturer’s instructions. The IEF was performed in a protein IEF cell (GE Healthcare) using a stepwise voltage gradient to 80 kVh. The strips were equilibrated for 2 × 15 min in equilibration buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris–HCl, pH 8.8) supplemented with 1% DTT and 4% iodoacetamide prior to running the second dimension. The SDS-PAGE was carried out vertically on 12.5% polyacrylamide gels using an Ettan DALT II system (GE Healthcare). Resolved proteins were routinely stained with Coomassie Brilliant Blue G-250 for protein identification purposes. All experiments were performed in triplicate. The reproducibility of the 2-DE was verified by running the same samples at least three times on separate gels. Three replicate gels from three independent experiments were run for each growth condition. The gels were compared using Image Master Platinum 5.0 software (GE Healthcare).
7
Two-Dimensional Gel Electrophoresis of Proteins
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Two-dimensional gel electrophoresis (2-DE) was performed according to the manufacturer's instructions (GE Healthcare, Milan, Italy) with slight modifications. Samples containing 0.125 mg proteins were in-gel rehydrated for 12 h onto 7-cm immobilized non-linear pH gradient strips (pH 3-10), electrofocused at 200 V, 500 V, and 1000 V each for 1 h (step-N-hold), 1000-5000 V (gradient) for 30 min, and 5000 V (step-N-hold) for 3 h using an IPGphor system (GE Healthcare).
The strips were then reduced for 30 min in SDS equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 1% (w/v) DTT, followed by 30 min alkylation in the same equilibration buffer with 2.5% (w/v) iodoacetamide instead of DTT. Electrophoresis was run on an Ettan DALT II system (GE Healthcare). The gels were stained with colloidal Coomassie Blue G250 (Bio-Rad, Hercules, CA, USA), scanned using image scanner and the gel images were analyzed with ImageMaster 2D Platinum 6.0 software (GE Healthcare).
The strips were then reduced for 30 min in SDS equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 1% (w/v) DTT, followed by 30 min alkylation in the same equilibration buffer with 2.5% (w/v) iodoacetamide instead of DTT. Electrophoresis was run on an Ettan DALT II system (GE Healthcare). The gels were stained with colloidal Coomassie Blue G250 (Bio-Rad, Hercules, CA, USA), scanned using image scanner and the gel images were analyzed with ImageMaster 2D Platinum 6.0 software (GE Healthcare).
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