Each hair protein solution of 100 μg was prepared for 2DE. In the conventional protocol, protein solution was mixed with rehydration solution which has similar constituent to the sample buffer prepared earlier, except a few grains of DTT was only added prior to use. A few grains of Orange G as tracking dye was also added. On the the other hand, hair protein solution was mixed with Destreak Rehydration solution (GE Healthcare, Amersham, UK) and 0.7 μL of IPG buffer (pH 3–10) in the modified protocol. Any undissolved substances were removed using a Spin Filter (Agilent Technologies, CA, USA). In both experiments, the volumes of protein solution added were based on the result of protein quantification. Moreover, different types of Immobiline DryStrips (13 cm, pH 4–7; 7 cm, pH 3–10) were also compared in this study. The former was adopted in the conventional protocol. In both experiments, the Immobiline DryStrips were incubated in the sample mixture on the Immobiline Drystrip Reswelling Tray (GE Healthcare, Uppsala, Sweden). Appropriate amount of Immobiline DryStrip Cover Fluid was added into the samples before the DryStrips were allowed to incubate for 18 h at room temperature (RT).
Destreak rehydration solution
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom
DeStreak Rehydration Solution is a laboratory product designed to rehydrate and prepare agarose gels for electrophoresis. It is used to restore the gel's moisture content and facilitate the even separation of biomolecules during the electrophoresis process.
Automatically generated - may contain errors
Lab products found in correlation
Ipg buffer, by GE Healthcare (4 mentions)
Immobiline drystrip, by GE Healthcare (4 mentions)
18 cm ipg drystrips, by GE Healthcare (3 mentions)
Ettan ipgphor3 ief unit, by GE Healthcare (2 mentions)
Protean 2 xi system, by Bio-Rad (2 mentions)
Ipgphor system, by GE Healthcare (2 mentions)
Ettan ipgphor 3 system, by GE Healthcare (2 mentions)
2 d clean up kit, by GE Healthcare (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Urea, by Merck Group (2 mentions)
Immobiline drystrip reswelling tray, by GE Healthcare (1 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions)
Nebuffer 3, by New England Biolabs (1 mentions)
Ettan daltsix, by Cytiva (1 mentions)
Multiphor 2, by Cytiva (1 mentions)
Nativepage novex 3 12 bis tris protein gels, by Thermo Fisher Scientific (1 mentions)
Ettan ipgphor isoelectric focusing system, by GE Healthcare (1 mentions)
Pdquest software, by Bio-Rad (1 mentions)
Ettan ipgphor 2 system, by GE Healthcare (1 mentions)
Typhoon 9400, by GE Healthcare (1 mentions)
29 protocols using destreak rehydration solution
1
Optimized 2DE Hair Protein Protocol
2
Phosphorylation analysis of XEE by 2D-electrophoresis
3
Two-Dimensional Protein Separation
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Soluble proteins were run in the first dimension using a commercial horizontal electrophoresis system (MultiphorII; Amersham Pharmacia Biotech). Then 100 μg of the protein sample were mixed with Destreak Rehydration Solution (GE Healthcare), dithiothreitol (DTT) (20 mM) and IPG buffer, pH 3–10 NL (GE Healthcare), and loaded onto ImmobilineTM DryStrip pH 3–10 NL, 24 cm (GE Healthcare). IPG strips were allowed to rehydrate overnight. Samples were run at 50 mA per strip. In the first step, voltage was ramped to 500 V during a 5-hour period, maintained at 500 V for another 5-hour period, re-ramped to 3500 V during a 9.5-hour period and was finally maintained at 3500 V for 5 h. After the first dimension, IPG strips were then equilibrated twice for 15 min in equilibration solution (0.05 M Tris–HCl, pH 8.8, 6 M urea, 30% v/v glycerol and 2% w/v SDS), first with 65 mM dithiothreitol (reduction step) and finally with 135 mM iodoacetamide (alkylation). The second dimension was done in a vertical electrophoresis system (Ettan DALTsix; Amersham Pharmacia Biotech) in a 12.5% (26 cm_20 cm_1 mm) polyacrylamide gel, where proteins were separated according to molecular size. The electrophoresis conditions were 1 W per gel until the dye front reached the bottom of the gel. Sets of three gels were used for each sampling time.
4
Phalotris mertensi Venom Proteome Analysis
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Phalotris mertensi venom sample (350 µg of proteins) was dissolved in MilliQ® water and mixed with DeStreak rehydration solution (GE Healthcare) containing 1% IPG buffer to a final volume of 450 µl (for 24 cm precast IPG strip; pH 3–10, linear) (GE Healthcare). First, dimension was carried out in an Ettan IPGphor Isoeletric Focusing System (GE Healthcare) as described by the manufacturer, at 20 °C, using a three-phase electrophoresis program: 30 V for 6 h, 150 V for 2 h, 350 V for 1 h, 500 V for 1 h, 1,000 V for 1 h, 3,000V for 1 h, and 5,000 V for 13 h. Prior to running the second dimension, the IPG strip was placed in a rehydration tray and the proteins were reduced and alkylated by sequential incubation in the following solutions: 0.05 M Tris-HCl, pH 8.4, 1% sodium dodecyl sulfate (SDS); 30% glycerol (equilibration buffer-EB), 20 mg/ml dithiothreitol in EB for 12 min; and then a solution of 30 mg/ml iodoacetamide in EB, for 10 min. Afterwards, it was directly applied onto a 12% SDS–polyacrylamide gel (20 cm × 26 cm × 1.5 mm) for the second dimension electrophoresis. The gel was stained with silver according to Mortz et al. (2001) (link).
5
Proteomic Analysis via 2D Gel Electrophoresis
6
Fractionation and Immunoblotting of Mitochondrial Proteins
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Isoelectrofocusing (IEF) was performed with 13-cm Immobiline DryStrips of 6–11L [linear] pH gradient using an Ettan IPGPhor3 IEF unit (GE Healthcare)18 (link). In brief, 200 µg of cellular protein diluted in 250 µl of rehydration buffer (DeStreak Rehydration Solution, GE Healthcare) containing 0.5% of the corresponding IPG buffer (GE Healthcare) were loaded in the 13-cm strips. The equilibrated strips were transferred to the top of a 12% SDS polyacrylamide gel. Electrophoresis was carried out using a Protean II XI system (Bio-Rad) with constant current (30 mA/gel) at 4 °C for 3 h. Western blot analysis of the fractionated proteins was performed as described above. For Blue Native (BN) gels, mitochondrial pellets were suspended in 50 mM Tris-HCl pH 7.0 containing 1 M 6-aminohexanoic acid at a final concentration of 10 mg ml−1. The membranes were solubilized by the addition of 10% digitonin (4:1 digitonin/mitochondrial protein). 5% Serva Blue G dye in 1 M 6-aminohexanoic acid was added to the solubilized membranes. Native PAGE™ Novex® 3–12% Bis-Tris Protein Gels (Life Technologies) were loaded with 70 μg of mitochondrial protein. After fractionation, the gels were electroblotted onto PVDF membranes. Membranes were further processed for immunoblotting.
7
2D-PAGE Analysis of B. lanceolatus Venom
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Precast IPG strips (13 cm, pH 3–10, linear, GE Healthcare) were rehydrated with 250 µL of DeStreak rehydration solution (GE Healthcare) containing 1% IPG buffer (GE Healthcare) and 100 µg of B. lanceolatus venom for 16 h at room temperature. First dimension separation was carried out using an Ettan IPGphor Isoelectric Focusing System (GE Healthcare), following th emanufacturer’s instructions. We followed a five-phase electrophoresis program; a step of 500 V for 60 min, a gradient of 1000 V for 60 min, a gradient of 8000 V for 2 h 30 min, a step of 1000 V for 12 h (so as to run overnight), and finally a step of 8000 V for 30 min. Right at the end of the separation, the IPG strips were sequentially incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 2% SDS, 6 M urea, 30% glycerol, bromophenol blue) containing 10 mg/mL DTT for 15 min and then in equilibration buffer containing 25 mg/mL iodoacetamide for 15 min. After that, they were briefly washed in electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) and applied to 12% SDS-polyacrylamide gels (10 × 10 cm) for second dimension electrophoresis, using the Hoefer Mini VE apparatus (GE Healthcare). The gels were finally silver-stained.
8
Proteomic Analysis of Sperm Cells
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Sperm cells (3 × 106) were washed twice with 9% (v/v) sucrose and solubilized with 250 μl of DeStreak® rehydration solution (GE Healthcare, Uppsala, Sweden) with the addition of 1% (v/v) IPG 3–11 solution (GE Healthcare, Uppsala, Sweden). This solubilization mixture contains optimized concentrations of urea, thiourea, CHAPS detergent, DeStreak® Reagent and ampholytes. It is a strong protein solubilizer that also prevents protein streaking and oxidation during the isoelectrophoretic run. Samples were processed for IEF by sonicating at amplitude 10 μm for 5 sec followed by centrifugation at 10 000 × g for 3 min, as previously described [34 (link)]. The particle-free supernatants after sonication were used for in-gel swelling of 13 cm long IEF strips pI 3–11 (GE Healthcare, Uppsala, Sweden). Focusing was performed on a Protean II IEF cell (BioRad, Hercules, CA, USA) up to 48 000 VXhr. Focused proteins were separated by placing the IEF strips on 14% SDS-PAGE gels (W160XL140X1.5 mm) along with 6–175 kDa markers. Electrophoresis was conducted so as to keep within gels proteins of molecular mass down to 6 kDa.
9
Quantitative Proteomic Profiling by 2D-PAGE
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In the experiment, 240 µg of total protein from each individual sample were diluted to up to 250 µL with DeStreak rehydration solution (GE Healthcare) and 0.5% 3–11 NL pH IPG buffer (GE Healthcare). Each mixture was loaded onto 13 cm IGP DryStrips (GE Healthcare) with a 3–10 NL pH gradient. The first-dimension separation—isoelectric focusing (IEF) —was then carried out on an Ettan IPGphor II system (GE Healthcare). IPG DryStrips were equilibrated in a reducing agent followed by an alkylating agent. The second dimension was performed by placing the strips on 8–16% CriterionTM TGX stain-free acrylamide gels (Bio-Rad) to allow protein separation by electrophoresis in a Criterion DodecaTM cell (Bio-Rad). The analytical gels were visualized with Typhoon 9400 (GE Healthcare) after SYPRO® (Bio-Rad) staining. The digitalized 2-DE gel images were studied (protein spot detection, spot matching and semi-quantitative statistical analysis) using PDQuest software (Bio-Rad). For each studied condition, three different gel images were analyzed and a corresponding reference synthetic image was obtained. To improve accuracy, detected spots and spot matches after in silico matching, were manually edited.
10
Two-Dimensional Gel Electrophoresis Protocol
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For two-dimensional gel electrophoresis, the two samples to be run on the same gel plus the internal standard were mixed before adding 2 × GE lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 12 µ ml−1 DeStreak reagent (GE Healthcare)) and 2% (v/v) ampholytes immobilized pH gradient (IPG) buffer (pH 3–10 NL, GE Healthcare) to a final volume of 125 µl. Isoelectric focusing (IEF) was carried out on pH 3–10 IPG-strips (24 cm, nonlinear gradient; GE Healthcare) using the IPGphor system from GE Healthcare. Immobiline DryStrips were rehydrated overnight with DeStreak rehydration solution (GE Healthcare) before cup-loading of proteins and IEF on an Ettan IPGphor Manifold (GE Healthcare). The migration was performed at 20°C (60 V for 2 h; gradient from 60 to 500 V for 5 h; hold 500 for 1 h, gradient from 500 to 1000 for 3 h; hold 1000 V for 1 h; gradient from 1000 to 8000 V for 4 h, hold 8000 V until 64 000 Vh). After the IEF, IPG strips were equilibrated twice for 15 min in equilibration buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue) supplemented with DTT and then with iodoacetamide. Second-dimension SDS–PAGE was performed using 24 cm format 12.5% resolving gel and run at 20°C overnight with 1.5 W per gel, using the Ettan DALT twelve system (GE Healthcare).
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