Chloroform/methanol-precipitated protein pellets from phosphorylation reactions were solubilized in rehydration buffer [7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 0.5% IPG buffer, 0.002% Bromophenol Blue and 1 mM DTT]. Proteins were applied to 11 cm immobilized DryStrip gels (pH 3–8; non-linear; GE Healthcare) and separated using the Bio-Rad Laboratories Protean IEF Cell Isoelectric Focusing system following the manufacturer’s instructions. Afterwards, the strips were equilibrated consecutively in equilibration buffer [75 mM Tris/HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 0.002% Bromophenol Blue] containing 10 mg/ml DTT and in equilibration buffer containing 25 mg/ml iodacetamide for 20 min each. Proteins were separated in the second dimension by SDS/PAGE (10% gel) and analysed as described above.
Drystrip gels
Manufactured by GE Healthcare
DryStrip gels are a type of laboratory equipment used for protein separation and analysis. They provide a stable and consistent platform for isoelectric focusing, a key technique in two-dimensional gel electrophoresis. The DryStrip gels are pre-cast, ready-to-use, and available in a range of pH gradients and lengths to accommodate various sample types and separation requirements.
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Lab products found in correlation
3 protocols using drystrip gels
1
2D Gel Electrophoresis of Phosphorylated Proteins
2
Proteomic Profiling of Gastric Cancer Cells
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Proteins were separated using 2-DE, as reported in our previous study (17 (link)). Briefly, 400 µg of protein per sample (equal quantity) were immobilized onto 18 cm (pH 4–7) DryStrip Gels (GE Healthcare Immobiline™; Amersham Biosciences) for the first dimensional electrophoresis (isoelectric focusing: IEF) using Ettan DALT II system (Amersham Biosciences). After the isoelectric focusing, the strips were equilibrated twice; first time with equilibration buffer containing 10 mg/ml dithiothreitol (DTT) and the second time with equilibration buffer containing 40 mg/ml iodoacetamide (IAA) for 15 min each. Proteins in equilibrated strips were then separated, depending upon the molecular weight, with the second dimension on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Silver nitrate staining methods were implemented as reported previously (18 (link)) with slight changes, and gels were prepared in triplicates for each assay condition. The silver-stained gels were scanned for image analysis, and Progenesis Samespots software version 4.0 Nonlinear Dynamics Ltd.) was used to perform spot density-based image analysis. Those spots were considered for further analysis depending on the difference in the spot intensities with fold-change ≥1.5 and statistical significance of P<0.05 in SCU-treated AGS and SNU484 cells, compared with the untreated (DMSO) groups.
3
Quantitative Proteomic Analysis via 2-DE
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Two-dimensional electrophoresis (2-DE) of protein samples was performed as described previously (Li et al., 2018 (link)). A protein sample of 1300 μg was loaded by rehydration to immobilize DryStrip gels (pH = 3–10 linear, 24 cm) (GE Healthcare, Waukesha, WI, United States) individually. After automated detection and matching, manual editing was carried out to correct the mismatched and unmatched spots. Spots were considered reproducible when they were well resolved in the three biological replicates. For each matched spot, a measurement was carried out for each biological replicate, and normalized volumes were computed using the total spot volume normalization procedure of the software. The normalized volume of each spot was assumed to represent its expression abundance. A criterion of p < 0.05 and an abundance ratio of at least 1.5 were used to define significant differences when analyzing parallel spots between different groups with two-way ANOVA.
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