2D gel electrophoresis was performed as previously described (Liu et al., 2015 (link)). Total proteins from Kc cells were extracted with NP-40 lysis buffer as described above. The protein extracts from each treatment were precipitated with ice-cold trichloroacetic acid/acetone and were then redissolved in rehydration buffer (8 M urea, 4% 3-[(3-Cholamidopropyl)-dimethyl- ammonio]-1-propane sulfonate [CHAPS], 65 mM DTT, 0.2% ampholytes, and 0.001% bromophenol blue). Equal amounts of the total proteins (150 μg) from each sample were loaded onto an immobilized pH gradient (IPG) strip with the pH ranging from 3 to 10 (Bio-Rad). Isoelectric focusing (IEF) was performed using an Ettan IPGphor 3 IEF System (GE Healthcare) following the manufacturer’s instructions. After IEF, the IPG strips were successively equilibrated in a reducing solution (0.375 M Tris-HCl, pH 8.8, 2% DTT, 6 M urea, 2% SDS, and 20% glycerin) and an alkylating solution (0.375 M Tris-HCl, pH 8.8, 2.5% iodoacetamide, 6 M urea, 2% SDS, 20% glycerin) each with 15 min. The equilibrated strips were then subjected to 2D SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes for probing with an antibody against biotin.
Ettan ipgphor 3 ief system
Manufactured by GE Healthcare
Sourced in United Kingdom
The Ettan IPGphor 3 IEF System is a laboratory instrument used for isoelectric focusing, a technique in protein separation. It provides a controlled environment for the separation of proteins based on their isoelectric point. The system includes a focusing unit, power supply, and software for method development and data analysis.
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Lab products found in correlation
Immobiline drystrip, by GE Healthcare (4 mentions)
Ipgbox, by GE Healthcare (2 mentions)
Ipg strip, by Bio-Rad (1 mentions)
Ettan dalt six apparatus, by GE Healthcare (1 mentions)
2 d clean up kit, by GE Healthcare (1 mentions)
Immobiline dry strips ph 3 10 nl, by GE Healthcare (1 mentions)
Sypro ruby, by Bio-Rad (1 mentions)
Ettan daltsix electrophoresis unit, by Cytiva (1 mentions)
Versadoc mp 4000 imaging system, by Bio-Rad (1 mentions)
Alphaimager gel documentation unit, by Bio-Techne (1 mentions)
Ipg strip, by GE Healthcare (1 mentions)
Ettan dalt 2 system, by GE Healthcare (1 mentions)
Coomassie brilliant blue r 250, by Carl Roth (1 mentions)
Immobiline drystrips ph 3 10, by GE Healthcare (1 mentions)
Biodocanalyze, by Analytik Jena (1 mentions)
Sonoplus, by Bandelin (1 mentions)
Imagemaster 2d platinum software v 7, by GE Healthcare (1 mentions)
Typhoon fla 7000, by GE Healthcare (1 mentions)
Ettan dige imager, by GE Healthcare (1 mentions)
Image master 2d platinum software version 6, by GE Healthcare (1 mentions)
17 protocols using ettan ipgphor 3 ief system
1
2D Gel Electrophoresis of Kc Cell Proteome
2
Proteomic Analysis of A. chrysogenum
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Intracellular proteins were harvested from the mycelium of A. chrysogenum in the fermentation stage [60 ]. One-milligram protein samples were mixed with a thiourea rehydration solution (8 M urea, 2% CHAPS, 0.3% IPG buffer, 10 mM DTT, 0.002% bromophenol blue). A total of 450 μl of sample solution was loaded onto ReadyStrip Linear IPG strips (pH3-10 non-linear [NL], 24 cm; GE) at room temperature for 2 h (rehydration). Then, the strips were loaded onto an Ettan™ IPGphor™ 3 IEF System (GE). Proteins were focused at 20°C using the following program: 100 V, 3 h; 500 V, 2 h; 1000 V, 2 h; 3000 V, gradient to 2 h; 8,000 V, gradient to 8 h; 10,000 V, 5 h; 500 V, 5 h. The focused IPG strips were first equilibrated for 15 min with equilibrium bufferI(6 M urea, 50 mM Tris–HCl pH 8.8, 2% w/v SDS, 1% w/v DTT, 0.002% bromophenol blue). For the second equilibration, equilibrium bufferII (DTT was replaced with 2.5% iodoacetamide for the equilibrium buffer) was used. For the second-dimension polyacrylamide gel electrophoresis (SDS-PAGE), the equilibrium strips were placed onto 12.5% polyacrylamide in an Ettan Dalt Six apparatus (GE Healthcare) for 1 hour at 1watts/gel and then 2 watts/gel for 10 hours. Proteins were stained with Coomassie Blue G-250 (Amersco) for at least 3 h and destained in 10% (v/v) acetic acid until a clear background was obtained.
3
Two-Dimensional Gel Electrophoresis Protocol
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IEF was performed using the Ettan IPGphor-3 IEF system (GE Healthcare) with 13 cm (Immobiline DryStrip, pH 4–7; GE Healthcare) gel strips. Prior to rehydration, the precipitated proteins were treated using the 2-D Clean-up Kit (GE Healthcare) to remove contaminants that interfere with electrophoresis. IPG strips were rehydrated overnight at room temperature (RT) with rehydration solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.2% DTT, 0.5% IPG buffer (pH 4–7), and 0.002% Bromophenol blue. Each strip was loaded with 200 μg of protein and IEF was carried out at 20°C for 12 h (maximum voltage of 8,000 V, maximum current of 50 μA/IPG strip, total 28,000 Vh).
4
2D Electrophoresis of OMV Bp Proteins
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2DE was performed using the Ettan IPGphor 3 isoelecteric focusing system (GE Healthcare, USA). The incubation of the samples for 2DE was performed after sample preparation and IR-labeling. About 350 μg OMV Bp proteins was incubated overnight at laboratory temperature in DeStreak Rehydration solution (final volume = 250 μL) which contained 7 M urea, 2 M thiourea, 100 mM, 4% CHAPS, 0.2% carrier ampholyte, 0.0002% bromophenol blue, 4 mg of 50 mM dithiothreitol (DTT) and 1.5 μL of IPG buffer (pH 3-10 NL) (GE Healthcare). Immobiline Dry Strips pH 3-10 NL, 7 cm (GE Healthcare) were rehydrated with protein sample in an IPGbox (GE Health-care). Isoelectric focusing (IEF) was performed in an Ettan IPGphor 3 IEF system (GE Healthcare) according to the following conditions: 300 V for 30 min, 1000 V for 30 min, 5000 V for 90 min, finally 5000 V for 8 KVh, at 20 °C and a current limit of 50 μA per strip. After IEF, strips were equilibrated in 3 mL of equilibration buffer (150 mM Tris-HCl, pH 8.8, 6 M urea, 50% glycerol, 20% SDS, and bromophenol blue) with DTT (2%) for 15 min at RT, followed by 3 mL of equilibration buffer with iodoacetamide (2.5%) for 15 min at RT. The proteins were separated in second dimension on 10% SDS -PAGE in a vertical electrophoretic dual gel system and were visualized by Coomassie (R-250) staining method (13 ).
5
Proteome Profiling of Plasma and Muscle
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Here, we briefly summarise the 2-DE procedure as the procedure was described in detail earlier [10 (link),23 (link),50 (link)]. Proteomic analysis of depleted plasma samples and prepared muscle biopsies was carried out using Ettan IPGphor 3 IEF System (GE Healthcare, Buckinghamshire, UK) (first dimension) and Ettan DALTsix Electrophoresis Unit (Amersham, Pharmacia, Uppsala, Sweden) (second dimension). The fluorescent stain SYPRO Ruby (Bio-Rad Laboratories, Hercules, CA, USA) was applied to plasma protein gels, and silver stain was applied to muscle biopsy gels. The stained protein pattern was visualized using a charge coupled device camera (VersaDoc Imaging system 4000 MP, Bio-Rad). The PDQuest Advanced (v. 8.0.1, Bio-Rad) software was used to analyse and quantify the protein pattern. The amount of protein in a certain spot was assessed as background corrected optical density integrated over all pixels in the spot and expressed as integrated optical density (IOD). The parts per million (ppm) values for all proteins were generated and used for further statistical analysis.
6
Separation of Phospholipase A2 Isoforms
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The protein fractions were submitted to electrofocusing in 13 cm strips with pHs ranging from 3 to 10 in a nonlinear form, using an Ettan IPGphor 3 IEF System (GE Healthcare). At the end of the electrofocusing, the strips containing the PLA2s were equilibrated and transferred to a 12.5% polyacrylamide gel where they were separated according to molecular mass. The electrophoretic run was performed at 25 mA and 100 W during 5.5 hours and at the end of the protein separation the gel was stained with Coomassie Brilliant Blue (CBB) G-250 and images of the gel were captured using Image Scanner (Amershan Bioscience).
7
Two-Dimensional Protein Separation Protocol
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110 μg of protein samples were loaded on the Ettan IPGphor 3 IEF system (GE Healthcare Life Science), and first-dimension electrophoresis was performed using 13 cm Immobiline DryStrip pH 3–10 (maximum voltage: 6700 v). The strips were then mixed with 65 mM dithiothreitol and 135 mM iodoacetamide and transferred onto a vertical gel (12 % SDS-PAGE, Hoefer SE600; GE Healthcare Life Science) for second-dimension separation, and finally visualized with Silver staining. Targeted gel spots were excised, destained, reduced at 60 °C with 10 mM dithiothreitol for 45 min, then cysteine-blocked at 25 °C with 55 mM iodoacetamide for 30 min. Samples were digested with trypsin at 37 °C for 16 h, and the resulting peptides were vacuum-dried for later use.
8
Two-Dimensional Proteome Analysis of UV-B Stress
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The first dimension isoelectric focusing (IEF) was performed using Immobiline DryStrip (pH 4–7, 13 cm; GE Healthcare Bio-Sciences AB, Sweden). Protein sample (500 μg) dissolved in rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.3% (w/v) DTT, and 0.5% (v/v) IPG buffer) was loaded onto each IPG strip and rehydrated overnight at 4°C in the dark. After rehydration, IEF was performed using the Ettan IPGphor 3 IEF system (GE Healthcare, UK) using the following steps; 2 h at 100 V, 2 h at 200 V, 2 h at 500 V, 2 h at 1500 V, 2 h at 3000 V, 2 h at 6000 V, and 8000 V until a total of 60000 Vh was reached. Following the two steps of equilibrations (Nouwens et al., 2002 (link)), the second dimension separation was carried out on 12% SDS-PAGE gel at 20 mA/gel using SE 600 Ruby multiple gel-electrophoresis system (GE Healthcare, UK). Three gels were run for each protein sample of UV-B treated and untreated control cultures. Gels were stained with Colloidal Coomassie Blue G-250 (Candiano et al., 2004 (link)) and images were captured using the AlphaImager Gel Documentation unit (Alpha Innotech, USA).
9
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.
10
Phosphoprotein Analysis Using 2D-PAGE
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An Immobiline Dry strip (pH 3–10 in the thalamus or pH 4–7 in the hippocampus and frontal lobe, 24 cm length, GE Healthcare) was rehydrated with 400 μg of phosphoprotein in 450 μl of rehydration buffer containing 4% CHAPS, 7 M urea, 2 M thiourea, 20 mM Trizma base, 65 mM DTT, 1% IPG buffer and 0.002% bromophenol blue for 14 hr at room temperature. Isoelectric focusing (IEF) was performed using the Ettan IPGphor 3 IEF System (GE Healthcare) for a total of 70 kVh. For the second dimension, SDS-PAGE was performed using an Ettan DALTsix Large Vertical system (Amersham, USA) according to the following procedures: 45 min at a constant power of 5 watts followed by 20 watts per gel until the bromophenol blue reached the bottom of the gel. The gels were then stained with 0.12% w/v Coomassie Brilliant Blue G250. The 2D gels were analyzed with the DeCyder software package (GE Healthcare, USA).
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