Protein samples were mixed with an equal volume of UTC buffer (6M urea, 3M thiourea, 8% CHAPS, 100 mM DTT, and 2% IPG buffer (GE, Amersham), and incubated for 30 min in ice. The content was then diluted to the required volume using rehydration buffer (7M urea, 2M thiourea, 4% CHAPS, 0.5% ampholytes, 50 mM DTT, 1% IPG buffer (GE, Amersham), and 0.004% bromophenol blue). The strips (IPG Strips-pH 3-10NL) were then focused in IPGphor III after 16 h of passive rehydration. Consecutively, the strips were subjected to reduction and alkylation. For reduction, the strips were incubated in SDS-equilibration buffer I (6 M urea, 50 mM Tris-Cl, 30% glycerol, 2% SDS, 0.004% bromophenol blue, and 1% DTT) for 15 min in a gel rocker. For alkylation, the strips were incubated in SDS-equilibration buffer II (6 M urea, 50 mM Tris-Cl, 30% glycerol, 2% SDS, 0.004% bromophenol blue, and 2.5% iodoacetamide) for 15 min in a gel rocker. The strips were then placed on top of 12% polyacrylamide gel and sealed with overlay of 0.5% agarose solution. The electrophoresis conditions were 0.5 W for 45 min and 2 W for 5–6 h until the tracking dye reached the bottom of the gel plate. After electrophoresis, the gels were stained according to Dyballa and Metzger.[20 ] Digital images of 2D-gels were acquired using ChemiDoc™ XRS imaging system (Bio-Rad) with internal calibration.
Ipg buffer
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom
IPG buffer is a specialized solution used in isoelectric focusing (IEF) electrophoresis techniques. It maintains a stable pH environment to facilitate the separation and analysis of proteins based on their isoelectric points.
Automatically generated - may contain errors
Lab products found in correlation
Chaps, by GE Healthcare (10 mentions)
Dithiothreitol (dtt), by GE Healthcare (6 mentions)
Immobiline drystrip, by GE Healthcare (5 mentions)
Urea, by GE Healthcare (4 mentions)
Destreak rehydration solution, by GE Healthcare (4 mentions)
2 d quant kit, by GE Healthcare (4 mentions)
Sodium dodecyl sulfate (sds), by GE Healthcare (3 mentions)
Trypsin, by Promega (3 mentions)
Ipg strip, by GE Healthcare (3 mentions)
Thiourea, by GE Healthcare (2 mentions)
Glycerol, by GE Healthcare (2 mentions)
Iodoacetamide, by GE Healthcare (2 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Readystrip ipg strip, by Bio-Rad (1 mentions)
Readyprep 2 d cleanup kit, by Bio-Rad (1 mentions)
Formic acid, by GE Healthcare (1 mentions)
Dl dithiothreitol, by GE Healthcare (1 mentions)
Hplc grade, by Thermo Fisher Scientific (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
Ipgphor isoelectric focusing system, by Cytiva (1 mentions)
59 protocols using ipg buffer
1
Two-Dimensional Protein Separation Protocol
2
2D-PAGE Protein Purification and Separation
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Excess salts and other impurities were removed from each sample set using a Ready Prep 2D Clean Up kit (Bio-Rad, Hercules, CA, USA) as per the manufacturer’s protocol, and protein concentrations were measured using a Quick Start Bradford Dye Reagent Protein Assay kit (Bio-Rad). For 2D-PAGE (2D polyacrylamide gel electrophoresis), 50 μg of protein samples were used to rehydrate 11-cm, pH 5–8 Ready Strip™ IPG strips (Bio-Rad) overnight in rehydration buffer: 8 M urea, 2 M thiourea, 2% CHAPS, 1.5 M Tris-HCl (pH 8.8), BPB, 0.35% dithiothreitol (DTT), 0.5% IPG buffer (pH 3–10; GE Healthcare, Pittsburgh, PA, USA). Rehydrated strips were subjected to IEF by using an Electrophoresis Power Supply EPS 3501 XL (GE Healthcare) at 20 °C throughout focusing, and then at 250 V for 15 min, 3500 V for 2 h 50 min, 3500 V for 12 h 57 min, and 500 V for 15 min, after which the strips were equilibrated (30 min) in equilibration buffer (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, with BPB). SDS-PAGE was conducted with 7.5% gels, and proteins were stained with Coomassie Brilliant Blue (CBB) or by silver staining.
3
Protein Extraction and Separation
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Urea, DL-dithiothreitol, iodacetamide, IPG buffer, and formic acid were purchased from GE (USA); SDS (sodium dodecyl sμlfonate), Tris (Tris(hydroxymethyl)aminomethane), TCA (Trichloroacetic acid), AP (Ammonium persμLfate) and tetramethylethylenediamine were purchased from Amresco (USA); TEAB (triethylammonium bicarbonate) and Coomassie Brilliant Blue G-250 were purchased from Sigma (USA); trypsin was purchased from Promega (USA); acetonitrile, HPLC grade and H2O were purchased from Thermo (USA).
4
2D-Gel-Based Raf1 Protein Analysis
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Cells were treated with Compound C and infected in 15 cm dishes, then 2-D gel samples were collected by scraping cells in 250 ul of 2-D rehydration buffer (4% CHAPS, 8 M urea, 0.5% IPG Buffer (pH 3.0-10.0; GE Healthcare), 0.002% Bromophenol Blue, 40 mM DTT (added fresh before extraction)). Samples were spun to pellet insoluble fraction and loaded on a 13-cm Immobline Drystrip isoelectric-focusing gel (pH 3.0-10.0; GE Healthcare), and proteins were separated in the first dimension using the IPGphor isoelectric-focusing system (Amersham Pharmacia Biotech) following manufacturer’s protocol. Then gel was equilibrated for 15 minutes in SDS equilibration buffer (6 M urea, 75 mM Tris-HCl (pH 8.8), 30% Glycerol, 2% SDS, 0.002% Bromophenol Blue), and proteins were run in the second dimension by SDS-based electrophoresis on a 10% polyacrylamide gel. Finally, samples were treated as a typical western blot, by transferring the proteins to a nitrocellulose membrane, blocking with milk, and immunoblotting for Raf1 as described above.
5
Moringa oleifera Seeds Antifungal Activity
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Moringa oleifera seeds were collected from trees at the Campus do Pici of the Federal University of Ceará – UFC (Ceará, Brazil) under authorization (number: 47766) of the Chico Mendes Institute for Conservation of Biodiversity – ICMBio. A voucher specimen (N EAC34591) was deposited in the Prisco Bezerra Herbarium (UFC). The yeasts C. albicans (ATCC 10231), C. parapsilosis (ATCC 22019), C. krusei (ATCC 6258), and C. tropicalis (clinical isolate) were obtained from the culture collection of the Laboratory of Emergent and Reemergent Pathogens – LAPERE, Department of Pathology and Legal Medicine, UFC. Rabbit blood was obtained from animals of colonies maintained at the UFC. Human blood samples (ABO system) were obtained from healthy donors in the blood bank HEMOCE (Hemotherapy Center of Ceará). Experimental protocols were approved by the Ethics Committee of UFC, Brazil (protocol number: 77/2016).
Molecular mass markers, chromatographic matrices, IPG buffer, and immobilized pH gradient gel strips were obtained from GE Healthcare Life Sciences (New York, NY, United States). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States).
Molecular mass markers, chromatographic matrices, IPG buffer, and immobilized pH gradient gel strips were obtained from GE Healthcare Life Sciences (New York, NY, United States). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, United States).
6
Protein Extraction and 2D Separation
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Proteins were precipitated and washed using the 2-D Clean-Up kit (GE Healthcare Bio-Sciences AB). Air-dried protein pellets were solubilized in sample solution containing 7 M urea, 2 M thiourea, 25 mM DTT, 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) and 2% IPG-Buffer (GE Healthcare Bio-Sciences AB, Upsalla, Sweden). Equal amounts of radioactivity (106 dpm) were loaded onto the gel in the first dimension. Isoelectric focusing was carried out using pH 4 to 7 Immobiline Dry Strips on a Multiphor II electrophoresis system (GE Healthcare Bio-Sciences AB). Two-dimensional separation was performed according to our standardized procedure [22 (link)].
7
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.
8
Isolation and Analysis of Plant Cell Wall Components
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The cell
wall materials were isolated following the method of Li et al.18 (link) The cellulose and lignin as well as proto-pectin
contents were measured with the method described by Zhao et al.19 (link) The experiments were performed in triplicate,
and the results were expressed as means. Analysis of variance (ANOVA)
was performed with Tukey’s test (P < 0.01)
for locating significant differences between mean values using SPSS
16.0 software (SPSS Inc., Chicago, IL, U.S.A.). Total soluble proteins
were isolated from 300 mg of the inflorescence stem of P.
lactiflora. Proteins were precipitated and purified following the procedures
described by Yu et al.21 In brief, proteins
were precipitated with 10% (w/v) trichloroacetic acid (TCA)–acetone
and recovered by centrifugation. The protein pellet was washed three
times with 100% acetone and then dissolved in the protein solubilization
buffer. The insoluble fraction was removed via centrifugation at 12 000g for 45 min, and a 0.5% (v/v) IPG buffer (pH 4–7)
from GE Healthcare (Piscataway, NJ, U.S.A.) was added into the soluble
protein fraction. Protein concentrations were quantified according
to the Bradford method.22 (link)
wall materials were isolated following the method of Li et al.18 (link) The cellulose and lignin as well as proto-pectin
contents were measured with the method described by Zhao et al.19 (link) The experiments were performed in triplicate,
and the results were expressed as means. Analysis of variance (ANOVA)
was performed with Tukey’s test (P < 0.01)
for locating significant differences between mean values using SPSS
16.0 software (SPSS Inc., Chicago, IL, U.S.A.). Total soluble proteins
were isolated from 300 mg of the inflorescence stem of P.
lactiflora. Proteins were precipitated and purified following the procedures
described by Yu et al.21 In brief, proteins
were precipitated with 10% (w/v) trichloroacetic acid (TCA)–acetone
and recovered by centrifugation. The protein pellet was washed three
times with 100% acetone and then dissolved in the protein solubilization
buffer. The insoluble fraction was removed via centrifugation at 12 000g for 45 min, and a 0.5% (v/v) IPG buffer (pH 4–7)
from GE Healthcare (Piscataway, NJ, U.S.A.) was added into the soluble
protein fraction. Protein concentrations were quantified according
to the Bradford method.22 (link)
9
CRC Tumor Protein Extraction Protocol
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All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by The North Denmark Region Committee on Health Research Ethics (Project identification No. 2-16-4-0001-03) and the Danish data agency. This project was further approved (18 December 2017) by the National Committee on Health Research Ethics (Project identification No. 60819). Biopsies were obtained from ten patients with CRC, Table 1 . From each patient 3 samples were collected, one from the central part, one from the peripheral part of the tumor, as well as one from the resection margin furthest from the tumor. The tissue samples were collected from the tumor and bowel just after the specimen was removed from the abdomen, rinsed in cold saline, frozen in liquid nitrogen, and stored at −80 °C. For protein extraction, initially, the tissue was rinsed with PBS-buffer if presence of excess blood was observed, then solubilized in IP 3-10 NL lysis buffer (9M Urea, 2% (vol/vol) Triton X-100, 2% (wt/vol) DTT, 2% (vol/vol) IPG-buffer (GE Healthcare)) using a homogenizer. Protein concentration was determined using the Non-Interfering Protein Assay (488250, Calbiochem®, Merck KGaA, Darmstadt, Germany).
10
Two-Dimensional Gel Electrophoresis for Protein Analysis
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After protein labelling, samples were prepared for the isoelectric focusing (IEF) step. Strip gels of 24 cm and a linear pH range of 4–7 (GE Healthcare) were used. Strips were initially rehydrated with labelled protein samples (7 M urea, 2 M thiourea, 2% CHAPS (w/v), 0.5% IPG buffer (v/v) (GE Healthcare), 2% DTT). Strips were then processed using an Ettan IPGPhor IEF system (GE Healthcare) in a total of 35,000 Volts.h-1 and, subsequently, reduced and alkylated for 30 min under slow agitation in Tris-HCl solution (75 mM), pH 8.8, containing 2% SDS (w/v), 29.3% glycerol (v/v), 6 M urea, 1% DTT (w/v) and 2.5% iodocetamide (w/v). The 2D- DIGE conditions were performed as described by Weiss and Görg [32 ]. Gels were immediately scanned with a FLA-9000 modular image scanner (Fujifilm Lifescience, Dusseldorf, Germany). To ensure maximum pixel intensity between 60,000 and 90,000 pixels for the three dyes, all gels were scanned at a 100 μm resolution and the photomultiplier tube (PMT) voltage was set between 500 and 700 V. As described by Agapito-Tenfen et al. [2 (link)], we performed preparative gels for each plant variety in order to extract relevant spots. These were performed with a 450 μg load of total protein pools in 24 cm gels from each variety, separately, and stained with colloidal Coomassie Brilliant Blue G-250 (MS/MS compatible).
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