High-resolution 2-DE was performed according to Görg et al. [43 (link)] with some modifications as previously described by Franco et al. [41 (link)]. The first dimension was run in 24 cm long pH 4–7 linear gradient ReadyStripTM IPG Strips (Bio-Rad Laboratories, Hercules, CA, USA), loaded with 450 µg of protein from each sample, concurrently with 0.6% DTT and 1% immobilized pH gradient (IPG) buffer (Bio-Rad Laboratories). Isoelectric focusing (IEF) was performed on PROTEAN IEF Cell (Bio-Rad Laboratories). Initially, 50 V were applied for 12 h to rehydrate each strip, following a voltage grade until 70 kWh. Subsequently, strips were immersed in equilibration buffer I (50 mM Tris pH 8.8, 30% glycerol, 2% SDS, 6 M urea and 1% DTT) for 15 min at room temperature and in the same conditions with equilibration buffer II (50 mM Tris pH 8.8, 30% glycerol, 2% SDS, 6 M urea and 2.5% iodoacetamide). For the second dimension, proteins were resolved on 13% SDS-PAGE gels of 24 × 20 cm using Ettan DALTsix vertical system (GE Healthcare).
Ipg buffer
Manufactured by Bio-Rad
Sourced in United States
IPG buffer is a laboratory solution used in isoelectric focusing, a technique for separating and analyzing proteins. The buffer provides a stable pH environment to facilitate the separation of proteins based on their isoelectric points. It is a critical component in the sample preparation process for two-dimensional gel electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Protean ief cell, by Bio-Rad (1 mentions)
Readystrip ipg strip, by Bio-Rad (1 mentions)
Ettan daltsix vertical system, by GE Healthcare (1 mentions)
Cyanine3 (cy3), by GE Healthcare (1 mentions)
Immobilized ph gradient strips, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Alanine aminotransferase, by Nanjing Jiancheng (1 mentions)
Aspartate aminotransferase, by Nanjing Jiancheng (1 mentions)
Bradford kit, by Sangon (1 mentions)
Ipg strip, by Bio-Rad (1 mentions)
Urea, by AppliChem (1 mentions)
Protease inhibitor, by Bio-Rad (1 mentions)
Chaps, by AppliChem (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Tetrahydrofuran thf, by Merck Group (1 mentions)
5 protocols using ipg buffer
1
High-Resolution 2-DE Proteomic Profiling
2
DIGE-Based Proteome Profiling
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All steps were operated in dark room. The proteome samples were labelled for DIGE analysis using Cy2, Cy3 and Cy5 CyDye™ DIGE Fluor minimal dye (GE Healthcare), respectively, according to the manufacturer manual (GE Healthcare). Cy2 was used to label an internal standard which was pooled equal amounts of each of all samples. Each 50 μg protein sample was labelled at a ratio of 400 pmol of dye on ice for 30 min, and the labelling reaction was terminated by adding 1 μl of 10 mM lysine and left on ice for 15 min. The three labelled samples were mixed into a single tube, and then both of extra 300 μg paired protein samples in a gel were added to the same tube, thus total of 750 μg protein samples were mixed in the tube and later could be used as preparative gel for spots picking. Equal volumes of 2× sample rehydration buffer (7 M urea, 2 M thiourea, 2% DTT, 4% CHAPS, 1% pH 3–10 NL IPG buffer (GE Healthacare), and 0.004% bromphenol blue) was added to the protein samples. Rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% DTT, 0.5% IPG buffer, and 0.004% bromphenol blue from Bio-Rad) was added to reach volumes to 450 μl for rehydration.
3
Protein Profiling of Liver Injury
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Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Hyaluronic acid (HA), laminin (LN), collagen III (Col III) and collagen IV (Col IV) ELISA kits were obtained from Abbott Laboratories, USA. A Bradford kit was bought from Sangon Biotech, Shanghai, China. Sirius red and hematoxylin-eosin (HE), protease inhibitor cocktail, 3-[(3-Cholamidopropyl) dimethylammonio]-propanesulfonate (CHAPS), glycine, ammonium persulfate (APS), TEMED, and protease inhibitor cocktail were obtained from Sigma Chemical (St. Louis, MO, USA) Dithiothreitol (DTT), glycine, Immobilized pH gradient (IPG) strips, IPG buffer, dry strip cover fluid and other reagents that were used in two-dimensional gel electrophoresis were acquired from Bio-Rad (Hercules, CA, USA).
4
2-DE Analysis of IgG Fractions
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We performed 2-DE of the IgG fractions as described previously [25] (link). A total of 20 µg of IgG, h-IgG or hs-IgG in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.2% DTT and 3.4 µl of immobilized pH gradient [IPG] buffer, pH 3–10) was used to rehydrate the IPG strip (7 cm, pH 7–10, Bio-Rad, USA) for 16 h. Isoelectric focusing (IEF) was performed at a constant temperature of 20°C using a continuous increase in voltage (up to 4000 V) for 65,000 Vh. Prior to the second dimension, the IPG strip was incubated for 15 min in equilibration buffer (20% [w/v] glycerol, 2% SDS, 2% DTT, 0.375 M Tris-HCl, pH 8.8) then further equilibrated for 15 min in equilibration buffer containing 2.5% iodoacetamide instead of 2% DTT. The strip was placed onto a 10% SDS-PAGE gel. Molecular weight markers were loaded onto a filter paper and placed next to the IPG strip. Low-melting point agarose was used to cover the IPG strip and filter paper. Proteins were separated using the same conditions described above for SDS-PAGE analysis of human IgG fractions.
5
Extracellular Vesicle Isolation Protocol
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One and a half million AF-MSCs and HPL cells were cultured in their specific media, as it is described in the previous section. At 80% confluency the media were removed and the cell layers were washed 3 times with 1xPBS (Gibco BRL, Grand Island, NY) and 1 time with serum and phenol red free medium (SFM) (Gibco-Invitrogen, Grand Island, New York). Cells were then incubated with SFM for 24 h and then conditioned media (CM) were collected as previously described [28 (link)]. The latter was centrifuged at 1000 ×g for 10 min at 4 °C to remove dead cells and large debris and incubated with 7.5% Trichloro Acetic Acid (TCA) (Fluka, Buchs Switzerland), 0.1% NLauroyl Sarcosine (NLS) (Fluka) at −20 °C overnight. Centrifugation then followed at 10,000 ×g for 10 min at 4 °C. The supernatant was discarded, and the pellet was washed in ice cold Tetra Hydro Furan (THF) (Sigma-Aldrich Ltd.) and centrifuged again as previously. The final pellet was air dried and resuspended in sample buffer [7 M urea (Applichem, Darmstadt, Germany), 2 M thiourea (Fluka), 4% CHAPS (Applichem), 1% DTE(Sigma), 2% IPG buffer (BioRad) and 3.6% Protease inhibitors (BioRad)] followed by 30 min bath sonication. Samples were stored at −80 °C until use.
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