Plasma exosomes were extracted using the magnetic bead sorting method. Briefly, 1 ml plasma was mixed with 5 ml magnetic bead (Stainless Steel Beads, Qiagen, Germany). After being washed for three times, exosomal proteins were lysed using Lysis Buffer 3 containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM ethylenediaminetetraacetic acid (EDTA). After vortexing (vortex-genie 2, Shanghai, China) and standing for 5 min, dithiothreitol (DTT) was added into the mixture to final concentration of 10 mM. The mixture was then centrifuged at 25,000g for 20 min at 4°C, and the supernatant was collected. After being incubated at 56°C for 1 h, the proteins were precipitated by adding four times volume of cold acetone and incubating at room temperature for 45 min in the dark. After centrifugation at 25,000g for 20 min, the pellets were collected and resuspended in X buffer. The concentration of plasma exosomal proteins was measured using the Bradford assay (Bio-Rad, Hercules, CA). The integrity quality control of the collected proteins was verified by protein electrophoresis on 12% uniform SDS-polyacrylamide gels using the Ettan DALT II system (Amersham Bioscience, Uppsala, Sweden) at 120 V for 120 min. The separated proteins were visualized by Coomassie blue staining.
Ettan dalt 2 system
Manufactured by Cytiva
Sourced in Sweden
The Ettan DALT II system is a protein separation instrument designed for two-dimensional electrophoresis (2-DE) of proteins. It is capable of high-resolution protein separation and analysis, providing researchers with a tool for proteomics studies.
Automatically generated - may contain errors
Lab products found in correlation
Bradford assay, by Bio-Rad (2 mentions)
Stainless steel bead, by Qiagen (1 mentions)
Ipg strip, by Cytiva (1 mentions)
Imagequant software, by GE Healthcare (1 mentions)
Drystrip cover fluid, by Cytiva (1 mentions)
Image scanner, by Cytiva (1 mentions)
2d quantification kit, by Cytiva (1 mentions)
Drystrip gels, by GE Healthcare (1 mentions)
6 protocols using ettan dalt 2 system
1
Plasma Exosome Extraction Protocol
2
Proteome Separation and Analysis by 2D-PAGE
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(i)1st dimension: separation by isoelectrofocalization (IEF). IEF separates proteins according to their isoelectric point (IP). IEF separation was performed on strips (IPG-strips, Amersham) with a PH gradient of 4 to 7. Then, the strips were rehydrated with 450 µl of rehydration buffer covered with mineral oil to avoid dehydration (Drystrip Cover Fluid, Amersham). Proteins were then isoelectrofocused at 30V for 3 h, then at 30V to 1000 V for 5 h, then from 1000V to 8,000 V for 4 h, and finally to 8,000 V for 5 h before coming back to 30 V again. (ii) 2nd dimension: SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis). SDS-PAGE separates proteins according to their molecular weight (MW). After IEF, the proteins contained in the strips were equilibrated in a buffer containing SDS (Sodium Dodecyl Sulfate), allowing the proteins to be negatively charged, so that they have the same electric charge and MW is the only parameter acting in SDS-PAGE. Gels were then set in a tank (Ettan Dalt II system, Amersham). Electrophoresis was performed at 20 mA/gel overnight at 4°C.
Gels were then revealed by using a scanner (Typhoon Variable Mode Imager 9400). For each gel, pictures were checked by the ImageQuant software (GE Healthcare Life Sciences).
Gels were then revealed by using a scanner (Typhoon Variable Mode Imager 9400). For each gel, pictures were checked by the ImageQuant software (GE Healthcare Life Sciences).
3
Proteome Profiling by 2D-PAGE
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The “mixed protein samples” (200 µg) were mixed with rehydration buffer and applied to an 18 cm linear IPG strips, pH 3–10 (Amersham Biosciences, Sweden).Then the strips were subjected to isoelectric focusing in IPGphor (Amersham Biosciences, Sweden). The focused strips were subsequently reduced with 1% DTT and alkylated with 2.5% iodoacetamide (IAM) in equilibrated buffer (6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl, pH 8.8). The treated strips were transferred to 12% SDS-PAGE in Ettan DALT II System (Amersham Biosciences, Sweden) for the secondary electrophoresis. The analytical gels were stained with silver staining without addition of glutaraldehyde. The stained gels were scanned using an Imagescanner (Amersham Biosciences, Sweden), and the images were analyzed using ImageMaster 2-D Platinum version 3.0 (Amersham Biosciences, Sweden).
4
Spinal Cord Proteomics after Nerve Injury
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The L4-5 spinal cord samples of animal tissues (Sham and CCI-treated rats) were prepared at day 7 after CCI and 2DGE and gel scanning were performed as previously described (Zou et al., 2012 (link)). The removed proteins were counted with a 2-D Quantification kit (Amersham Biosciences, Sweden). The proteins of spinal cords were separated with 2DGE as previously described (Zou et al., 2012 (link)). Briefly, each protein sample (600 μg) was diluted and put on IPG strips (pH 3–10 NL, 24 cm) for 14 h of rehydration at 30 V. The proteins were divided based on the pI under the state (1 h at 500 V, 1 h at 1,000 V, and 8.5 h at 8,000 V to assemble 68 kVh) on an IPGphor (Amersham Biosciences). Aimed proteins in IPG strips were equilibrated for 15 min in a mixture (6 M of urea, 2% SDS, 30% glycerol, 50 mM of Tris-HCl, pH 8.8, and 1% DTT), and alkalized for another 15 min in a mixture (6 M of urea, 2% SDS, 30% glycerol, 50 mM of Tris-HCl, pH 8.8, and 2.5% iodoacetamide). The aimed proteins in the IPG strips were separated based on the Mr with SDS-PAGE (12%) on the Ettan DALTII system (Amersham Biosciences). The Coomassie blue G-250 staining was implemented to view the 2DGE-separated proteins. We pull all the lumber spinal cord protein samples of each group together. 2DGE of each group was done in triplicate.
5
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.
6
Plasma Exosome Isolation and Characterization
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Plasma exosomes were extracted using the magnetic beads sorting method. Brie y, 1 ml plasma was mixed with 5 ml magnetic bead. After washed for three times, exosomal proteins were lysed using Lysis Buffer 3 containing 1 mM phenylmethylsulfonyl uorid (PMSF) and 2 mM ethylene diamine tetraacetic acid (EDTA). After votexing (vortex-genie 2, Shanghai, China) and standing for 5 min, dithiothreitol (DTT) was added into the mixture to nal concentration of 10 mM. The mixture was then centrifuged at 25,000 g for 20 min at 4ºC and the supernatant was collected. After incubated at 56 ºC for 1 h, the proteins were precipitated by adding four times volume of cold acetone and incubating at room temperature for 45 min in the dark. After centrifugation at 25,000 g for 20 min, the pellets were collected and resuspended in X buffer. The concentration of plasma exosomal proteins were measured using the Bradford assay (Bio-Rad, Hercules, CA). The integrity quality control of the collected proteins was veri ed by protein electrophoresis on 12% uniform SDS-polyacrylamide gels using the Ettan DALT II system (Amersham Bioscience, Uppsala, Sweden) at 120 V for 120 min. The separated proteins were visualized by Coomassie blue staining.
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