Proteins were extracted from CSF samples using a 2D cleanup kit (GE, 80-6484-51), resuspended in 100 μl of lysis buffer (30M Tris pH 8.5, 7M Urea, 2M Thiourea, 4% CHAPS) and quantitated by Bradford assay using BSA as a standard. Cy-dye labeling, isoelectric focusing and gel electrophoresis of 2D-cleaned CSF samples were performed according to standard DIGE protocols.31 (link) Labeled samples were combined (1 Cy3, 1 Cy5 and 25 μg of Cy2 sample/gel) and diluted 2X with rehydration buffer [7M Urea, 2M thiourea, 2% CHAPS, 1% pH 3-10 IPG buffer (GE Healthcare), 50mM DTT, 1% saturated bromophenol blue solution] to a final volume of 450 μL and then isoelectric focused on an IPGphor II (GE Healthcare) instrument using standard isoelectric focusing protocols for pH 3-10 strips (GE Healthcare). Finally, pH strips were reduced and alkylated, placed in 20x24cm 12% SDS-PAGE gels, and run in a Dalt-12 electrophoresis system (GE Healthcare) at 2 watts per gel for 45 minutes, followed by 15 watts per gel until the dye front reached the bottom (~4 hours).
Ipgphor 2
Manufactured by GE Healthcare
Sourced in United States
The IPGphor II is a laboratory instrument designed for isoelectric focusing (IEF), a technique used in protein analysis and separation. It provides a stable, controlled environment for the separation of proteins based on their isoelectric points. The IPGphor II is capable of processing up to 12 samples simultaneously, with a temperature range of 0-99.9°C and a maximum voltage of 10,000V.
Automatically generated - may contain errors
Lab products found in correlation
2 d clean up kit, by GE Healthcare (2 mentions)
Ph 3 10 strips, by GE Healthcare (1 mentions)
Ipg buffer ph 3 10, by GE Healthcare (1 mentions)
Imagescanner 2, by GE Healthcare (1 mentions)
2 d quant kit, by GE Healthcare (1 mentions)
Ettan daltsix electrophoresis system, by GE Healthcare (1 mentions)
Imagemaster 2d platinum software version 7, by GE Healthcare (1 mentions)
Anti myc tag antibody, by Cell Signaling Technology (1 mentions)
Pharmalyte 3 10, by GE Healthcare (1 mentions)
Immobiline drystrip, by GE Healthcare (1 mentions)
9 protocols using ipgphor 2
1
Proteomic Profiling of Cerebrospinal Fluid
2
Two-Dimensional Electrophoresis Protocol for Proteome Analysis
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To perform a 2DE, 130μg of cell proteins extract were solubilized using isoelectrofocusing buffer (IEF). The buffer contains 4% CHAPS, 8 M urea, 0.1 M dithiothreitol (DTT), 0.8% pH 3–10 nonlinear (NL) carrier ampholyte buffer. IEF was performed at 70,000 Vh, on the IPGphor II apparatus (GE Healthcare, Chicago, IL, USA), using nonlinear Immobline Dry Strips (GE Healthcare), pH 3–10, 24 cm long. After this first dimension, the strips were equilibrated with SDS equilibration buffer containing DTT 10 mg/mL−1, for 15 min and then for another 15 min in SDS equilibration buffer with iodoacetamide (IAA)25 mg/mL−1. Procedures were performed according to GE Healthcare Ettan protocol book [16 (link),17 ]. The second dimension was carried out on 10% SDS polyacrylamide gels, until the bromophenol blue reached the bottom of the gels [18 (link)]. The gels were fixed and stained using silver staining method, which is compatible with mass spectrometry analysis [19 (link)].
For each sample, the analysis was carried out in triplicate. Gel images were acquired using Image Scanner II (GE Healthcare, Chicago, IL, USA) and analyzed through Image Master 2D-Platinum software 6.0 version (GE Healthcare, Chicago, IL, USA)
For each sample, the analysis was carried out in triplicate. Gel images were acquired using Image Scanner II (GE Healthcare, Chicago, IL, USA) and analyzed through Image Master 2D-Platinum software 6.0 version (GE Healthcare, Chicago, IL, USA)
3
Proteomic Analysis of M. tuberculosis
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Protein extracts were obtained from 140 mL of M. tuberculosis mid-log cultures grown in 7H9–0.05% Tween 80 supplemented with 10% ADC. Cells were pelleted by centrifugation and washed twice with sterile PBS 1X. Pellets were resuspended in lysis solution (7M Urea, 2 M Thiourea, 4% CHAPS, 4% ASB-14) and then sonicated (20 cycles: 30 sec ON/1 min OFF) at 4 °C in ice water, and finally centrifuged to remove cells debris. Samples were then quantified and purified using 2-D Quant Kit and 2-D Clean-Up Kit (GE Healthcare) respectively. For two-dimensional gel electrophoresis (2DE) IPGphor II and EttanSix (GE Healthcare) were used for the first and second dimension respectively. 100 μg of each sample was separated by 2DE in 24 cm length, 6–9 pH strips in the first dimension and in a 12% SDS-PAGE for the second dimension according to manufacturer instructions. 2DE gels were silver stained as described elsewhere31 (link). Image analysis was performed using Progenesis SameSpot software (Nonlinear Dynamics). After image analysis, differential spot was excised and identified as previously described by mass spectrometry32 (link).
4
2D Gel Electrophoresis of Proteins
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Total proteins (500 μg) were loaded onto GE Healthcare 24 cm IPG gel strips (pH 4–7). The isoelectric focusing (IEF) of the acidic range IPG strips (pH 4–7) was carried out according to the manufacturer’s instructions of IPGPhor II (GE Healthcare, USA) at 20 °C for a total of 65 kVh40 (link). Two-dimensional SDS-PAGE gels (12.5% linear gradient) were ran on an Ettan Daltsix electrophoresis system (GE Healthcare, USA). The procedure was set as 2.5 W per gel for 30 min, followed by 12 W per gel for 4–5 h. After electrophoresis, the protein gels were stained for spot detection using silver nitrate, as previously described41 (link).
Gel images were analyzed using the Imagemaster 2D Platinum Software Version 7.0 (GE Healthcare). Spot detection was carried out using the software with the values of the parameters smooth, minimum area, and saliency set to 2, 15, and 8, respectively. Manual spot editing, such as spot deletion, splitting, and merging was performed. The determined relative spot intensities were subsequently used for statistical analysis38 (link).
Gel images were analyzed using the Imagemaster 2D Platinum Software Version 7.0 (GE Healthcare). Spot detection was carried out using the software with the values of the parameters smooth, minimum area, and saliency set to 2, 15, and 8, respectively. Manual spot editing, such as spot deletion, splitting, and merging was performed. The determined relative spot intensities were subsequently used for statistical analysis38 (link).
5
Isoelectric Focusing of Protein Samples
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IPG strips with a non-linear pH gradient from 3–11 and a length of 24 cm (GE Healthcare) were rehydrated overnight in 450 μl TUC1% (6 M Urea, 2 M Thiourea, 50% ACN, 1% CHAPS, 0.5% Pharmalyte 3–10, 200 mM HED), samples were applied to the IPG strips via anodic cup-loading and IEF was performed according to published protocols31 (link) on a IPGphor II (GE Healthcare) in darkness. The IEF was stopped after 27,000 Vhrs.
6
Proteome Profiling of Hippocampal Samples
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The 2D electrophoresis was performed as previously reported and repeated for three times [18 ]. Briefly, hippocampal samples containing 300 μg of protein were loaded per tube in an isoelectric focusing system (IPGphor II, GE). The samples were isoelectrofocused and separated with sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). The gels were silver-stained. The protein spots that either increased or decreased for more than twofold were selected for matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) identification. The mascot software package and the database of SwissProt were used to match the mass of peptides [23 (link)].
7
Two-Dimensional Gel Electrophoresis of Nuclear Proteins
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Two-dimensional gel electrophoresis (2-DE) was performed using IPGphor II (GE Healthcare) as previously described (Cozzolino et al., 2013 (link)). In brief, proteins (90 µg) from nuclear extracts were precipitated with 100% acetone and then loaded on pH 3–10 IPG strips (IPGs) and electrofocused at 15,000 V/h at a maximum voltage of 5,000 V. The second-dimension separation was performed at a constant current of 50 mA for 2 h. Proteins were transferred to nitrocellulose membranes (Protran Nitrocellulose Transfer Membrane, Schleicher & Schuell, BD Biosciences), and Western blot was performed as described above with mouse monoclonal anti-Myc-Tag antibody (9B11, 1:1,000; Cell Signaling Technologies Inc. Danvers, USA).
8
Comprehensive 2D-PAGE Protein Analysis
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Eighteen 24 cm immobilized pH gradient (IPG) strips with a pH range of 3-10NL (Cat. No. 1632043, Bio-rad, Hercules, California, USA) were rehydrated in 500 μl of TUC1% (6 M urea, 2 M thiourea, 1.2% (v/v) 2,2 Dithiodiethanol (Cat. No. 380474, Sigma-Aldrich), 0.5% (v/v) Pharmalyte 3-10 (GE Healthcare) overnight at room temperature, then stored at −80°C until further use. 5μg of protein sample mixed with 5 μg IPS was loaded via anodal cup-loading. Isoelectric focusing (IEF) was carried out using an IPGPhor II (GE Healthcare) with following settings: 150 V for 1 h, 300 V for 1 h, 600 V for 1.5 h, increase to 8,000 V by gradient over 2 h, 24.000 Vh at 8,000 V, under exclusion of light. Current was limited to 50 μA per IPG strip. After IEF, IPG strips were stored at −80°C until further use. SDS-PAGE was carried out as described earlier (12 (link)).
9
Avenin Characterization by IEF-SDS-PAGE
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For further characterization of avenins, two biological extracts with two replicates per extract were used. Isoelectric focusing (IEF) was performed using the IPGPhor II apparatus (GE Healthcare) on 13 cm Immobiline dry strips of 3–10 linear pH gradients. Passive re-hydration was performed overnight in a solution containing 7 M urea, 2 M thiourea, 70 mM DTT, 1% IPG buffer (pH 3–10), 4% CHAPS, 0.34% anti-protease, and 100 µg (analytical gel) or 1 mg (preparative gel) of the protein extract. IEF was carried out by applying a cumulative voltage of 30 and 60 kVh for analytical and preparative gel, respectively.
Following IEF, proteins were reduced for 15 min in an equilibration buffer containing 0.05 M Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 1% DTT, followed by alkylation for 15 min in the same buffer containing 2.5% iodoacetamide instead of DTT. The second dimension was performed using SDS–PAGE gels (12% T, 2.1% C) sealed with 0.5% agarose in SDS buffer on Hoefer vertical system (GE Healthcare). The migration conditions were 10 mA/gel for first 30 min then 35 mA/gel until the exit of the dye front. Gels were stained with CBB.
Following IEF, proteins were reduced for 15 min in an equilibration buffer containing 0.05 M Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS, and 1% DTT, followed by alkylation for 15 min in the same buffer containing 2.5% iodoacetamide instead of DTT. The second dimension was performed using SDS–PAGE gels (12% T, 2.1% C) sealed with 0.5% agarose in SDS buffer on Hoefer vertical system (GE Healthcare). The migration conditions were 10 mA/gel for first 30 min then 35 mA/gel until the exit of the dye front. Gels were stained with CBB.
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