Ipg buffer ph 3 10

Manufactured by GE Healthcare

Sourced in Sweden, United Kingdom

IPG buffer pH 3–10 is a ready-to-use buffer solution used in isoelectric focusing (IEF) techniques. It establishes a stable pH gradient from 3 to 10 that allows the separation and analysis of proteins based on their isoelectric points.

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Lab products found in correlation

2 d quant kit, by GE Healthcare (2 mentions) 2 d clean up kit, by GE Healthcare (2 mentions) Agilent 3100 offgel fractionator, by Agilent Technologies (1 mentions) Readyprep 2 d cleanup kit, by Bio-Rad (1 mentions) Pellet pestle, by Merck Group (1 mentions) Protease inhibitor, by GE Healthcare (1 mentions) Chaps, by GE Healthcare (1 mentions) Modified trypsin, by Promega (1 mentions) Urea, by Scharlab (1 mentions) Ultrapure grade water, by Merck Group (1 mentions) Ethylene diamine tetraacetic acid (edta), by Scharlab (1 mentions) Protease inhibitor cocktail p8340, by Merck Group (1 mentions) Sucrose, by Merck Group (1 mentions) Ammonium bicarbonate, by Merck Group (1 mentions) Bradford protein assay kit, by Bio-Rad (1 mentions) Immobiline drystrip, by GE Healthcare (1 mentions) Sep pak tc18 1 cc vac cartridge, by Waters Corporation (1 mentions) Immobiline drystrip ph 3 10, by GE Healthcare (1 mentions) Destreak, by GE Healthcare (1 mentions) Ipgphor 3, by GE Healthcare (1 mentions)

11 protocols using ipg buffer ph 3 10

Peptide Fractionation and Purification

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To increase proteome coverage of the haptenated peptide digests, the samples were separated into 12 fractions based on their isoelectric points. This was performed using the Agilent 3100 OFFGEL Fractionator in combination with 13 cm Immobiline IPG strips, pH 3-10. 100 µg peptide samples were made up to a final volume of 1.4 ml with a 1: 50 solution of IPG buffer, pH 3-10 (GE Life Sciences, Buckinghamshire, UK) diluted in 5% Glycerol. Peptides were focused for 20 kVh at a maximum current of 50 µA and a maximum power of 200 mW.
Each fraction was collected and acidified by adding 10 µl of solvent containing 10% TFA. Each acidified fraction was loaded onto a conditioned C₁₈ reverse-phase Empore Plates (3 M, Maplewood, Minnesota, USA), and washed with 20 µl of 0.5% acetic acid. Peptides were eluted from the tip using 40 µl of 80% acetonitrile + 0.5% acetic acid. Samples were lyophilized using a vacuum concentrator to 6 µl and mixed with 6 µl of 2% acetonitrile + 1% TFA.

Parkinson E., Aleksic M., Cubberley R., Kaur-Atwal G., Vissers J.P, & Skipp P. (2017). Determination of Protein Haptenation by Chemical Sensitizers Within the Complexity of the Human Skin Proteome. Toxicological Sciences, 162(2), 429-438.

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Protein Extraction and Analysis from Fish Mucus

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For each mucus sample taken from individual fish from Trial#1 100 μL of protein extract was precipitated using the ReadyPrep 2-D Cleanup Kit (Bio-Rad Laboratories, Hertfordshire, UK) following the manufacturer’s guidelines. The final protein precipitate was solubilised in 100 μL re-swell buffer (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.3% dithiothreitol (DTT), 1% (v) IPG Buffer pH 3–10 (GE Healthcare). Complete solubilisation of the pellet was achieved by briefly vortexing the tubes followed by clarification by centrifugation at 14,500 x g for 15 minutes. The supernatant was then transferred to a clean 1.5 mL tube. The protein samples were initially analysed by 1-Dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie brilliant blue G-250 (CBB) (Fisher Scientific, Loughborough, UK) staining to determine the sample quality and to provide an estimate of the protein concentration.

Micallef G., Cash P., Fernandes J.M., Rajan B., Tinsley J.W., Bickerdike R., Martin S.A, & Bowman A.S. (2017). Dietary Yeast Cell Wall Extract Alters the Proteome of the Skin Mucous Barrier in Atlantic Salmon (Salmo salar): Increased Abundance and Expression of a Calreticulin-Like Protein. PLoS ONE, 12(1), e0169075.

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Proteomic Analysis of Ag/AgCl NP Intoxication in Aedes aegypti Larvae

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Intoxication treatment for proteomic analysis was performed by treating two-day-old A. aegypti larvae with the Ag/AgCl NPs at the LC₂₅ concentration. After 24 h treatment, the surviving larvae were cultured further for 24 h without Ag/AgCl NPs. The midgut of the surviving A. aegypti larvae was dissected. A. aegypti larvae without NP treatment were used as a negative control. Samples of 100 midguts were pooled in 100 µL of rehydration solution (8 M urea, 2% w/v CHAPS, 40 mM dithiothreitol, 0.5% v/v IPG buffer pH 3–10 (GE Healthcare Life Sciences, Uppsala, Sweden), 0.02% w/v bromophenol blue) containing protease inhibitors (Roche Diagnostics, Mannheim, Germany). Then, the midguts were homogenized on ice using a pellet pestle (Sigma-Aldrich, Saint Louis, MO, USA) and the extracted proteins were cleaned using a 2-D Clean-Up kit (GE Healthcare Life Sciences, Uppsala, Sweden), following the manufacturer’s instructions. Then, the protein pellets were solubilized in the rehydration solution and quantified using a 2-D Quant Kit (GE Healthcare Life Sciences, Uppsala, Sweden). The midgut protein concentration was adjusted to 50 μg in 120 μL of the rehydration solution.

Chimkhan N., Thammasittirong S.N., Roytrakul S., Krobthong S, & Thammasittirong A. (2022). Proteomic Response of Aedes aegypti Larvae to Silver/Silver Chloride Nanoparticles Synthesized Using Bacillus thuringiensis subsp. israelensis Metabolites. Insects, 13(7), 641.

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Macrophage Protein Extraction Protocol

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Protein extraction was performed as previously described [18 (link)], briefly; macrophages were washed in PBS thrice, to remove bovine protein contaminants, then collected and homogenised in protease inhibitor cocktail solution (CompleteMini; Roche, Switzerland). Supernatant was collected and protein precipitation, using 10% trichloroacetic acid in acetone, was performed at -20°C for 2 h. Samples were centrifuged at 4°C, 19800g for 10 min and supernatant discarded, remaining pellet was washed in 20 mM dithiothreitol in acetone and centrifuged as before and supernatant discarded. Samples were then reconstituted in 250 μL of urea sample solution, containing; 9 M urea, 1% dithiothreitol, 4% CHAPS, 2% IPG buffer pH 3–10 (GE Healthcare, UK) and 1% bromophenol blue. Aliquots were taken for protein concentration determined by 2-D Quant Kit (GE healthcare) performed to manufacturer guidelines.

Ward L.J., Ljunggren S.A., Karlsson H., Li W, & Yuan X.M. (2017). Exposure to atheroma-relevant 7-oxysterols causes proteomic alterations in cell death, cellular longevity, and lipid metabolism in THP-1 macrophages. PLoS ONE, 12(3), e0174475.

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Comprehensive Proteomics Sample Preparation

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LC-MS grade ACN, TFA, formic acid (FA), EDTA, Tris, glycerol, urea, thiourea and DTT were from Scharlab (Scharlab S. L., Barcelona, Spain). Ultrapure grade water was from Millipore (EMD Millipore Co., Billerica, MA, USA).
Sucrose, protease inhibitor cocktail (P8340) and ammonium bicarbonate were from Sigma-Aldrich (Sigma-Aldrich Co., St. Louis, MO, USA). Bradford Protein Assay Kit was from Bio-Rad (Bio-Rad, Hercules, CA, USA). Immobilized pH gradient gel (IPG) strips and IPG buffer pH 3-10 were from GE healthcare (GE healthcare, Uppsala, Sweden). Modified trypsin was from Promega (Promega, Madison, WI, USA).

Sentandreu E., Fuente-García C., Navarro J.L, & Sentandreu M.A. (2021). A straightforward gel-free proteomics pipeline assisted by liquid isoelectric focusing (OFFGEL) and mass spectrometry analysis to study bovine meat proteome. Food science and technology international = Ciencia y tecnologia de los alimentos internacional, 27(2).

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iTRAQ Fractionation and LC-MS/MS Analysis

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The iTRAQ mixture was fractionated using an Agilent OFFGEL 3100 fractionator with a 24 cm GE Healthcare Immobiline DryStrip with a nonlinear pH range of 3–10. The strip was rehydrated with a buffer containing 4.8% glycerol and 0.96% IPG buffer pH 3–10 (GE Healthcare) for 15 min. Peptides were diluted in 3.6 mL of the buffer and added in equal amounts to each well. Fractionation was achieved using the default program for peptides “OG24PE01” which targets 50 kVh with maximum values of 4500 V, 200 mW, and 50 mA. The 24 resulting fractions were combined into 18 fractions and desalted using a Sep-Pak tC₁₈ 1 cc Vac Cartridge (Waters). Each fraction was concentrated using a vacuum centrifuge and resuspended in 0.1% formic acid. Four microgram of labeled peptides were injected into the LC-MS/MS ((liquid chromatography tandem mass spectrometry).

de Oliveira Dal'Molin C.G., Orellana C., Gebbie L., Steen J., Hodson M.P., Chrysanthopoulos P., Plan M.R., McQualter R., Palfreyman R.W, & Nielsen L.K. (2016). Metabolic Reconstruction of Setaria italica: A Systems Biology Approach for Integrating Tissue-Specific Omics and Pathway Analysis of Bioenergy Grasses. Frontiers in Plant Science, 7, 1138.

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Two-Dimensional Electrophoresis of Mosquito Midguts

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The 2-DE was performed at the Proteomics and Protein Biochemistry Laboratory, Universidade Federal de Viçosa. Midguts were taken from each mosquito developmental stage, and three 2-DE gel (technical replicates) from a pool of 200 mosquitoes was done for each stage or feeding condition. Each Immobiline DryStrip pH 3-10, 7 cm (GE Healthcare) was hydrated for 16 h with 125 μL of a solution containing 85 μg protein added Destreak (GE Healthcare), 40 mM dithiothreitol (DTT), and 2% (v/v) IPG buffer pH 3-10 (GE Healthcare). Isoelectric focusing (IEF) was performed using the IPGphor3™ (GE Healthcare) system in accordance with the manufacturer's instructions.
After IEF, strips were reduced for 15 min in a 5 mL buffer solution containing 75 mM Tris-HCl pH 8.8, 1% DTT, 6 M urea, 30% glycerol (v/ v), 2% SDS (w/v), and 0.002% (w/v) bromophenol blue, and then alkylated in the same buffer by substituting DTT with 2.5% iodoacetamide. The second dimension was performed using polyacrylamide gel, 12% T glycine-SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis), according to Laemmli [10] . The proteins were visualized using Coomassie Brilliant Blue staining (CBB) G-250 [11] . The 2-DE gels were scanned at 300 dpi using Image Scanner III (GE Healthcare) and analyzed by Image Master III (GE Healthcare), which identifies and quantifies the intensity of % volume protein spot.

Fernandes K.M., de Magalhães-Júnior M.J., Baracat-Pereira M.C, & Martins G.F. (2016). Proteomic analysis of Aedes aegypti midgut during post-embryonic development and of the female mosquitoes fed different diets. Parasitology international, 65(6 Pt A).

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Comprehensive Protein Extraction and Analysis

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Dithiothreitol, iodoacetamide, ammonium persulfate, urea, thiourea, and pancreatin were purchased from Sigma-Aldrich Co., Ltd.; an immobilized pH gradient (IPG) Drystrip (24 cm), pH 3-10, and IPG buffer, pH 3-10, were bought from GE Healthcare; and a ProteoMiner kit was obtained from Bio-Rad.

Zheng R.J., Wu R.J, & Ma X.D. (2017). Serum proteomic spectral characteristics of acute myeloid leukemia and their clinical significance. Genetics and molecular research : GMR, 16(2).

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Protein Precipitation and Solubilization

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Proteins in the collected fluid were precipitated immediately by adding five volumes of cold 0.1 M ammonium acetate in methanol, containing 0.07% β-mercaptoethanol. Samples were kept overnight at -20ºC and then centrifuged at 20,000 xg for 20 min. The pellet was washed twice with cold methanol, dried with N 2 gas and solubilized in a sample rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, 50 mM DTT, 2 mM PMSF and 0.2% (v/v) IPG buffer pH 3-10 (GE Healthcare, Uppsala, Sweden). After rehydration, samples were incubated in a Thermomixer comfort device (Eppendorf AG, Hamburg, Germany) at 42 ºC and 1,000 rpm during 3 h, then centrifuged at 10,000 xg for 10 min at RT and filtered (0.45 µm ultrafree-MC filters, Millipore, Bedford, USA). Protein concentration in the samples was quantified immediately with the Bradford method using an Asys UVM 340 spectrophotometer with microtiter plates (Biochrom Ltd., Cambridge, UK) and BSA as standard.

Ceballos-Laita L., Gutierrez-Carbonell E., Takahashi D., Abadía A., Uemura M., Abadía J, & López-Millán A.F. (2018). Effects of Fe and Mn deficiencies on the protein profiles of tomato (Solanum lycopersicum) xylem sap as revealed by shotgun analyses. Journal of proteomics, 170.

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Proteomic Profiling of Cerebrospinal Fluid

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Proteins were extracted from CSF samples using a 2D cleanup kit (GE, 80-6484-51), resuspended in 100 μl of lysis buffer (30M Tris pH 8.5, 7M Urea, 2M Thiourea, 4% CHAPS) and quantitated by Bradford assay using BSA as a standard. Cy-dye labeling, isoelectric focusing and gel electrophoresis of 2D-cleaned CSF samples were performed according to standard DIGE protocols.^{31 (link)} Labeled samples were combined (1 Cy3, 1 Cy5 and 25 μg of Cy2 sample/gel) and diluted 2X with rehydration buffer [7M Urea, 2M thiourea, 2% CHAPS, 1% pH 3-10 IPG buffer (GE Healthcare), 50mM DTT, 1% saturated bromophenol blue solution] to a final volume of 450 μL and then isoelectric focused on an IPGphor II (GE Healthcare) instrument using standard isoelectric focusing protocols for pH 3-10 strips (GE Healthcare). Finally, pH strips were reduced and alkylated, placed in 20x24cm 12% SDS-PAGE gels, and run in a Dalt-12 electrophoresis system (GE Healthcare) at 2 watts per gel for 45 minutes, followed by 15 watts per gel until the dye front reached the bottom (~4 hours).

Martin-Vaquero P., da Costa R.C., Allen M.J., Moore S.A., Keirsey J.K, & Green K.B. (2015). Proteomic Analysis of Cerebrospinal Fluid in Canine Cervical Spondylomyelopathy. Spine, 40(9), 601-612.

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