Protein samples extracted from CAM explants were labeled using the CyDye DIGE Fluors (minimal dyes) for Ettan DIGE kit (GE Healthcare, UK) according to the manufacturer. To a pool containing 50 μg of an equal mixture of proteins extracted from infected and uninfected CAM explants produced from three fetuses, 400 pmol of dyes were added. Dye swap was performed and Cy2 dye was used as internal standard. Labeled samples and 800 μg of mixture of unlabeled proteins were incubated with the IPG strip (18 cm, pH 4–7—GE Healthcare) in a rehydration apparatus (Immobiline DryStrip Reswelling Tray, GE Healthcare, UK) for approximately 12 h and subjected to isoelectric focus using Ettan™ IPGphor™ 3 Isoelectric Focusing System (GE Healthcare, UK) and the program IPGphor (GE Healthcare, UK). Electrophoresis was performed in 12% polyacrylamide gel containing SDS in vertical electrophoresis apparatus (Ettan DALTsix Electrophoresis Unit—GE Healthcare, UK) and scanned using Typhoon Trio (GE Healthcare, UK) with excitation/emission wave lengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm). After scanning, gels were stained with Coomassie Brilliant blue G-250 (Thermo Scientific, USA) for marking the spots of interest.
Ipgphor
Manufactured by GE Healthcare
Sourced in Sweden, United States, United Kingdom
The IPGphor is a laboratory instrument used for isoelectric focusing, a technique in protein analysis. It provides a controlled environment for the separation and purification of proteins based on their isoelectric point. The device features a modular design and automated control, enabling precise and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Ipg strip, by GE Healthcare (3 mentions)
Cydye dige fluors, by GE Healthcare (2 mentions)
Protean 2 xl 2 d cell apparatus, by Bio-Rad (2 mentions)
13 cm ipg strips, by GE Healthcare (2 mentions)
Low mr electrophoresis calibration kit, by GE Healthcare (2 mentions)
Processor plus, by Cytiva (2 mentions)
Gd 2000 vacuum gel dryer system, by GE Healthcare (2 mentions)
Personal densitometer si, by Cytiva (2 mentions)
Immobiline drystrip reswelling tray, by GE Healthcare (1 mentions)
Ettan ipgphor 3 isoelectric focusing system, by GE Healthcare (1 mentions)
Ettan daltsix electrophoresis unit, by GE Healthcare (1 mentions)
Coomassie brilliant blue g 250, by Thermo Fisher Scientific (1 mentions)
Typhoon trio, by GE Healthcare (1 mentions)
Versadoc 4000mp system, by Bio-Rad (1 mentions)
Pd quest v8, by Bio-Rad (1 mentions)
Spss v21, by IBM (1 mentions)
Ettan daltsix large vertical system, by GE Healthcare (1 mentions)
Imagescanner 3 labscan 6, by GE Healthcare (1 mentions)
Ltq ft ultra mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Polybrene, by Shimadzu (1 mentions)
18 protocols using ipgphor
1
Differential Proteome Analysis of CAM Explants
2
Two-Dimensional Gel Electrophoresis for Macrophage Proteomics
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2-DE was performed horizontally using an IPGphor and Multiphor (GE Healthcare) setup according to a method previously described by Görg, et al. [19 (link)], used by our group in the analysis of macrophage cell samples [18 (link)]. Macrophage protein samples, approximately 150 μg of protein per sample, were separated by 2-DE, with the following experimental replicates: 5 untreated control; 5 2mix treated; 3 7-ketocholesterol treated; 3 cholesterol treated; and 3 ethanol treated. First dimension isoelectric focussing was performed on pH 3–10 non-linear IPG strips (GE Healthcare) for 46000 Vh. Second dimension was performed using home cast homogenous gels (SDS-PAGE; T = 14%, C = 1.5%) and run overnight. 2-DE gels were stained using silver nitrate, according to the protocol set by Shevchenko, et al. [20 (link)]. Stained gels were imaged using a charge-coupled device camera and VersaDoc 4000MP system (Bio-Rad Laboratories, CA, USA) and gels spots matched and quantified, as parts per million (ppm) of total gel density, between gels using PD-Quest v8.0.1 (Bio-Rad Laboratories). Statistical analysis of matched protein spots between control untreated cells and treatments; 2mix, 7-ketocholesterol, cholesterol and ethanol was performed using a non-parametric Mann-Whitney U test (SPSS v21; IBM, UK), where significance was set at p ≤ 0.05.
3
Two-Dimensional Electrophoresis of Proteins
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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).
4
Protein Sequencing and Mass Spectrometry Analysis
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A total of 200 µg protein was mixed with 250 µl rehydration solution and rehydrated for 14 h using an IPGphor (GE Healthcare, Beijing, China). The isoelectric focusing was achieved with four steps, then carried out by two-dimensional gel electrophoresis (19 ). Following pretreatment, the samples (50 pmol/20 µl) were directly sequenced using a Shimadzu PPSQ-31A automated gas phase protein sequencer (Shimadzu Corporation, Kyoto, Japan). Briefly, samples were dissolved in 20 µl CH3CN (37%; v/v) solution and applied to TFA-treated glass fiber membranes pre-treated with polybrene (Shimadzu Corporation). Data were recorded using Shimadzu PPSQ version 31A software (Shimadzu Corporation). An accurate molecular mass of SCH-P9 and SCH-P10 peptides was determined using an LTQ FT Ultra mass spectrometer (Thermo Fisher Scientific, Inc.). The 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were recorded in d-dimethyl sulfoxide (DMSO) on a Bruker AV-400 spectrometer (Bruker Corporation, Billerica, MA, USA) at working frequencies 400 and 100 MHz. Chemical shifts are expressed in parts per million (δ) values and coupling constants (J) in Hz. High resolution electrospray ionization mass spectrometry (HRESI-MS) were measured on the LTQ FT Ultra instrument. The above procedure was performed by Wuhan Moon Biosciences Co., Ltd. (Wuhan, China).
5
Multidimensional Protein Extraction and Analysis
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The experiment was performed according to previous protocols [52 (link)]. Proteins were extracted using a lysis buffer (8M urea, 4% CHAPS, 50mM dithioerythritol and 0,0002% Bromophenol blue) and rehydrated with 8M urea, 2% CHAPS, 20mM dithioerythritol, 0.8% IPG buffer, carrier ampholytes pH 6-11 linear. The first dimension isoelectric focusing (IEF) was performed in immobiline dry strips (GE) with a pH range from 7 to 4. IEF was performed on IPGphor (GE) according to the manufacturer recommendations. The gels were then equilibrated in 6M urea, 3% SDS, 375mM Tris pH 8.6, 30% glycerol, 2% DTE and then incubated with 3% iodoacetamide (IAA) and traces of bromophenol blue (BBP). The second dimension was performed using an 8% SDS-PAGE gel. Transfer and detection were carried out as previously described.
6
Two-Dimensional Gel Electrophoresis Protocol
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Cells were washed three times with ice-cold PBS, after which total cell lysates were prepared by sonication in lysis buffer (9 M urea, 2% CHAPS, 0.8% pharmalyte and 1% DTT). The lysates were centrifuged at 15,000 g for 30 min, after which the supernatant was collected and stored at −80 °C. Samples were applied to 18-cm long pH 4–7 IPG strips (GE Healthcare, Piscataway, NJ) for 12 h at 10 V using IPGphor (GE Healthcare). Isoelectric focusing was performed for 1 h at 500 V and for 1 h at 1,000 V, after which the voltage was gradually increased to 8,000 V for 1 h. After focusing at 8,000 V for an additional 4 h, each strip was incubated for 20 min in buffer containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% glycerol, 2% SDS and 1% DTT, and then for an additional 20 min in the same buffer containing 2.5% indoleacetic acid (IAA) instead of DTT. The equilibrated strip was placed onto 10–15% gradient polyacrylamide gel and the gel was electrophoresed at 70 V in running buffer (25 mM Tris-HCl, pH 8.8, 192 mM glycine and 0.1% SDS).
7
Two-Dimensional Gel Electrophoresis of Proteins
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Cell lysates were precipitated with TCA/acetone. Protein pellets were resuspended with rehydration buffer (7 M Urea, 2 M thiourea, 4% CHAPS, 2% DTT, 0.5% IPG buffer pH 4–7, 1X protease inhibitor cocktail [EMD Millipore Corp., Billerica, MA, USA], 1 mM Na3VO4). Afterward, 100 μg of each protein sample was loaded onto strip gels (7 cm, pH 4–7; GE Healthcare Life Sciences) and cover fluid (GE Healthcare Life Sciences) was added to the manifold cup-loading system. Samples were placed on IPGphor (GE Healthcare Life Sciences), rehydrated for 12 hours, and electrofocused. The separated strip gels were incubated in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M Urea, 2% SDS, 30% glycerol, 0.002% Bromophenol Blue) containing 10 mg/mL DTT for 15 minutes and then incubated with equilibration buffer containing 25 mg/mL iodoacetamide instead of DTT for 15 minutes. The equilibrated strip gels were applied to 1.0-mm thick 12% acrylamide gels and sealed with 0.8% (w/v) agarose solution. SDS-PAGE was carried out at 20 mA for 6 hours. The gels were stained by coomassie brilliant blue R-250 or assayed by Western blotting.
8
Two-Dimensional Protein Separation by IEF
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For IEF (isoelectricfocusing), 100 μg of each protein sample were loaded onto the strip gels, rehydrated for 12 h (18 cm, pH 4–7) with rehydration buffer (7 M urea, 2 M thiourea, 2% v/v CHAPS, 2% IPG buffer (pH 4–7). The protein samples were then electrofocused in a manifold cup-loading system with IPGphor (GE Healthcare, Piscataway, NJ, USA), and 2nd dimension was carried out at 15 mA overnight using a PROTEAN II xl 2-D Cell apparatus (BIO-RAD, Hercules, CA, USA) following our previous described procedure52 (link).
9
Isoelectric Focusing of Lipoprotein Lipase
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After protein precipitation, partially purified LPL was processed as described elsewhere (Casanovas et al., 2009a (link)) and applied to rehydrated immobilized pH-gradient (IPG) strips (11 cm, pH 6–11; GE Healthcare, Uppsala, Sweden) by cup-loading at the cathode. Isoelectric focusing (IEF) was performed at 20°C on IPGphor (GE Healthcare, Uppsala, Sweden) according to the following protocol: linear ramp to 500 V in 1 h, linear ramp to 1,000 V in 1 h, linear ramp to 5,000 V in 1 h and 5,000 V/h up to 25 kV h. After that, focused IPG strips were cut at 7.5 cm from the anode (pH 6) and equilibrated. Equilibrated strips were loaded onto a 9% w/v polyacrylamide gel and sealed using a solution containing 0.5% w/v agarose, 25 mM Tris, 0.1% w/v SDS, 192 mM glycine and bromophenol blue for protein separation in the second dimension by SDS-PAGE. SDS-PAGE was run until the blue dye front reached the bottom of the gel.
10
Two-Dimensional Protein Electrophoresis Protocol
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Samples of the cellular protein extracts prepared as described above were subjected to two dimensional-polyacrylamide gel electrophoresis (2D-PAGE). One hundred μg of each protein sample was loaded onto the strip gels and rehydrated for 12 h (18 cm, pH 4–7) with rehydration buffer (7 M urea, 2 M thiourea, 2% v/v CHAPS, 2% IPG buffer, pH 4–7) (GE Healthcare, Piscataway, NJ, USA). The samples were then electrofocused in a manifold cup-loading system with IPGphor (GE Healthcare, Piscataway, NJ, USA) in the following sequential steps; pre-separation at 100 V for 1 h, 200 V for 1 h, 500 V for 1 h; application of the samples to the strip gels initially at 1,000 V for 4 h; gradient focusing from 1,000 V to 8,000 V for 30 min; and finally steady-state focusing at 8,000 V for 8 h. The separated strip gels were equilibrated with equilibration buffer (6 M urea, 2% SDS, 50 mM Tris-Cl, pH 8.8, 30% glycerol) containing 65 mM DTT for 15 min. After a second equilibration with the same buffer containing 2.5% v/v iodoacetamide instead of DTT and a trace of bromphenol blue for 15 min; the equilibrated strip gels were applied to 1.0 mm thick 10% acrylamide gels and sealed with 0.25% (w/v) agarose. SDS-PAGE was carried out at 15 mA overnight using a PROTEAN IIxl 2-D Cell apparatus (BIO-RAD, Hercules, CA, USA).
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