The first dimension was performed in a PROTEAN IEF cell system (Bio-Rad, CA, USA). Protein extracts of 1 mg protein were loaded on immobilized pH 3–10 nonlinear gradient strips with a length of 17 cm (GE-Healthcare, Art.: 17-1235-01) and actively rehydrated with 300 μL rehydration buffer consisting of 6 M urea (Sigma), 2 M thiourea (Sigma), 2% CHAPS (Sigma), 16 mM DTT (Sigma), 0.5% Bio-Lyte Ampholytes pH 3 10 (Fluka) at 50 V for 12 h at 20°C.
Protean ief cell system
Manufactured by Bio-Rad
Sourced in United States
The Protean IEF cell system is a laboratory equipment used for isoelectric focusing (IEF), a technique that separates proteins based on their isoelectric point. The system provides a controlled environment for the IEF process, allowing for the separation and analysis of complex protein mixtures.
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Lab products found in correlation
Ipg strip, by Bio-Rad (4 mentions)
Readystrip ipg 7 cm strips, by Bio-Rad (2 mentions)
Readystrip ipg strip, by Bio-Rad (2 mentions)
Bio lyte 3 10 ampholyte, by Bio-Rad (2 mentions)
Mini protean tetra cell, by Bio-Rad (2 mentions)
Readyprep 2 d cleanup kit, by Bio-Rad (2 mentions)
Chaps, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
Thiourea, by Merck Group (1 mentions)
Silver stain plus, by Bio-Rad (1 mentions)
Clean up kit, by GE Healthcare (1 mentions)
Cb x protein assay kit, by G Biosciences (1 mentions)
Ettan daltsix large vertical electrophoresis system, by GE Healthcare (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Readyprep 2 d starter kit rehydration sample buffer, by Bio-Rad (1 mentions)
Readystrip, by Bio-Rad (1 mentions)
Coomassie blue g 250, by Bio-Rad (1 mentions)
Bromophenol blue, by Bio-Rad (1 mentions)
2 d clean up kit, by GE Healthcare (1 mentions)
22 protocols using protean ief cell system
1
Protein Extraction and Isoelectric Focusing
2
Albumin and IgG Depletion from Ascites
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High-abundance proteins (albumin and IgG) were removed from the ascitic fluid using Vivapure anti HSA/IgG for Human albumin and an IgG depletion kit (Sartorius Stedim Biotech, Cat. No. VS-P08HAIGG), according to the manufacturer’s protocol. Ascites fluids depleted from HSA/IgG (200 μg) were cleared of salts contamination with a ProteoExtract Protein Precipitation Kit (Calbiochem, Cat. No. 539180) as per manufacturer’s protocol. Pellets were processed as described [20 (link)]. Briefly, samples were diluted in rehydration buffer containing 8 M urea, 0.5% (w/v) CHAPS, 10 mM DTT, 0.001% bromophenol blue, and Bio-Lyte 3–10 Ampholyte (0.2%) (Bio-Rad, Cat. No.163-1113). The protein mixture was then applied to ReadyStrip™ IPG 7 cm strips, pH 5–8 (linear) (Bio-Rad, Cat. No. 163–2004). Rehydrated strips were isoelectrically focused using a PROTEAN IEF cell System (Bio-Rad, Cat. No. 165–4000). To perform the second dimension analysis, the strips were processed by 15% SDS-PAGE and a Protean II XL Cell System was used. Finally the 2D gels were stained with Silver Stain Plus (Bio-Rad, Cat. No. 161–0449).
3
Protein 2-DE Protocol for Separation
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Protein 2-DE was performed as previously described [47 (link)]. A total of 1,200 μg of proteins extracted from each sample were first separated by isoelectric focusing (IEF) using gel strips with a pH gradient of 4 to 7 (Immobiline Dry Strip, pH 4–7 NL, 17 cm; BioRad, Hercules, CA, USA). The strips were rehydrated for 14 h in 320 ml of dehydration buffer and then focused at 20°C for a total of 64 kV-h with a PROTEAN IEF Cell system (Bio-Rad). After IEF, the strips were equilibrated for 20 min, first in equilibration buffer I [6 M urea, 0.375 M Tris (pH 8.8), 2% (w/v) SDS, 20% (v/v) glycerol, and 2% (w/v) DTT) and then in equilibration buffer II [6 M urea, 0.375 M Tris (pH 8.8), 2% (w/v) SDS, 20% (v/v) glycerol, and 2% (w/v) iodoacetamide]. The equilibrated strips were then placed over 12.5% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels for 2-DE at 25 mA for 5 h. The 2-De gels were stained with colloidal CBB.
4
Two-Dimensional Electrophoresis of Meat Proteins
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Total protein extracts from lyophilized meat samples were obtained according to Franco et al. [35 (link)]. Extraction and protein purification were performed with the Clean-Up kit (GE Healthcare, Uppsala) from crude cell lysates obtained by ultrasonic disruption using a Branson digital sonifier (model S-250, Branson Ultrasonics, Danbury). Protein concentration in samples was assessed with an improved Bradford method using the CB-X protein assay kit (G-Biosciences, St. Louis) following the instructions of the manufacturer to remove interfering agents and use with a microplate reader. The bovine serum albumin (BSA) protein standard was used to determine protein concentration from calibration curves.
Proteins from lyophilized meat samples were separated by 2-DE as previously described by Franco et al. [35 (link)]. Briefly, 350 μg of each biological replicate were loaded onto an immobilized pH gradient (IPG) strip (24-cm long, pH 4–7 linear gradient, ReadyStrip IPG strips, Bio-Rad Laboratories, Hercules). First-dimension isoelectric focusing (IEF) of proteins in strip gel was performed using a PROTEAN IEF cell system (Bio-Rad Laboratories). The second dimension was run on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using an Ettan DALTsix large vertical electrophoresis system (GE Healthcare).
Proteins from lyophilized meat samples were separated by 2-DE as previously described by Franco et al. [35 (link)]. Briefly, 350 μg of each biological replicate were loaded onto an immobilized pH gradient (IPG) strip (24-cm long, pH 4–7 linear gradient, ReadyStrip IPG strips, Bio-Rad Laboratories, Hercules). First-dimension isoelectric focusing (IEF) of proteins in strip gel was performed using a PROTEAN IEF cell system (Bio-Rad Laboratories). The second dimension was run on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using an Ettan DALTsix large vertical electrophoresis system (GE Healthcare).
5
Comparative Proteomics of Arabidopsis Mutants
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One gram of total protein was isolated from Col-0, GSH-fed Col-0, GSH-fed myb21 and GSH-fed bzip10 mutant lines of Arabidopsis. The extracted protein was then resuspended in IEF buffer consisting of 7 M urea, 2 M thiourea, 4% CHAPS, 20 mM DTT and 1% (w/v) Bio-Lyte (3/10) ampholyte (Bio-Rad) as before. Quantification of protein was performed by using the Bradford method. Protein of approximately 600 μg was re-hydrated in an immobilized pH gradient strip (7 cm; pH 4–7; GE) for 12 h. Isoelectric focusing was performed as follows: 250 V for 30 min, 8000 V for 2 h, 8000 V for 26 000 V h, 750 V for 1 h on a Bio-Rad PROTEAN IEF Cell system. After 15 min equilibration of focused strips in calibration buffers I and II (Bio-Rad) second-dimension gels were run by using 12% SDS polyacrylamide gels and staining with colloidal Coomasie Brilliant Blue (CBB) G-250.
6
Two-Dimensional Gel Electrophoresis Workflow
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IEF was performed using the Bio-Rad-Protean-IEF-Cell-System. Twenty-four centimeter IPG strips (Bio-Rad, USA) pH 4–7 were passively rehydrated overnight with 750 μg of protein in 300 μl of solution containing 1% carrier ampholyte (Bio-lyte 4–7; Bio-Rad, USA). The total product time × voltage applied was 63,500 Vh for each strip at 20 °C. Strips were subsequently reduced (1%DTT, 15 min) and alkylated (2.5% IAA, 15 min) during the equilibration step (30 min in 50 mM Tris–HCl pH8.8, 6 M urea, 30% glycerol v/v, 1%SDS, bromophenol blue). Equilibrated strips were then placed on SDS-polyacrylamide gels, 22 cm × 26 cm, 15% acrylamide, and sealed with 0.5% agarose. Protein spots were stained by coomassie blue. To ensure protein pattern reproducibility, three technical replicates were done.
7
Proteome Analysis of Protein Oxidation
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The samples used (native and oxidized albumin) for the identification of carbonylated proteins were prepared for analysis by two-dimensional gel electrophoresis.
First, both native and oxidized albumin were precipitated with acetone overnight at −20°C. The following day, aliquots of solubilized samples containing 500 µg of proteins were mixed with sample buffer containing 30 mM DTT, 0.5% v/v ampholytes (Biolytes pH 3–10; Bio-Rad, Hercules, CA, USA), and 0.001% w/v bromophenol blue to a total volume of 300 µL and loaded on IPG strips (17 cm, pH range: 4–7; Bio-Rad) for protein electrofocusing in a Protean IEF cell system (Bio-Rad). Strips were passively rehydrated for 2 hours at 20°C, and then the voltage was gradually increased (30–8,000 V) till it reached 60,000 Vh.
Prior to the separation of proteins according to their molecular weight, strips were equilibrated for 20 minutes in equilibration buffer (6 M urea, 1.5 M Tris–HCl pH 8.8, 2% SDS, and 20% glycerol) containing 2% DTT, followed by 20 minutes in equilibration buffer containing 135 mM iodoacetamide. Second-dimension separation was performed in polyacrylamide Mini-Protean TGX precast gel (BioRad) using a Mini-Protean tetra cell (Bio-Rad). Gels were run at a constant current intensity of 60 mA during 3 hours. A total of two gels were performed for each condition from two different biological samples.
First, both native and oxidized albumin were precipitated with acetone overnight at −20°C. The following day, aliquots of solubilized samples containing 500 µg of proteins were mixed with sample buffer containing 30 mM DTT, 0.5% v/v ampholytes (Biolytes pH 3–10; Bio-Rad, Hercules, CA, USA), and 0.001% w/v bromophenol blue to a total volume of 300 µL and loaded on IPG strips (17 cm, pH range: 4–7; Bio-Rad) for protein electrofocusing in a Protean IEF cell system (Bio-Rad). Strips were passively rehydrated for 2 hours at 20°C, and then the voltage was gradually increased (30–8,000 V) till it reached 60,000 Vh.
Prior to the separation of proteins according to their molecular weight, strips were equilibrated for 20 minutes in equilibration buffer (6 M urea, 1.5 M Tris–HCl pH 8.8, 2% SDS, and 20% glycerol) containing 2% DTT, followed by 20 minutes in equilibration buffer containing 135 mM iodoacetamide. Second-dimension separation was performed in polyacrylamide Mini-Protean TGX precast gel (BioRad) using a Mini-Protean tetra cell (Bio-Rad). Gels were run at a constant current intensity of 60 mA during 3 hours. A total of two gels were performed for each condition from two different biological samples.
8
Identification of Bovine Lactoferrin-Binding Outer Membrane Proteins
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The outer membrane proteins were cleaned using Ready Prep™ 2-D cleanup kit (Bio-Rad), and proteins were dissolved in Ready Prep™ 2-D starter kit Rehydration/sample buffer (Bio-Rad); this sample was used to passively rehydrate the immobilized pH gradient (IPG) ReadyStrips (7 and 17 cm, linear, pH 3–10; Bio-Rad) during 16 h at 20 °C. Isoelectrofocusing (IEF) of the proteins was run in a Protean IEF Cell System (Bio-Rad) in the following steps: 250 V for a 20 min linear ramp, 250 V for a 1 h rapid ramp, 500 V for a 1 h rapid ramp, 400 V for a 2 h linear ramp, and 4000 V with a rapid ramp up to 10 000 V-h. After reduction and alkylation in the equilibration buffer, IPG strips were subjected to separation by MW on 12% SDS-PAGE. The gel was transferred onto a nitrocellulose membrane and processed for overlay with the different proteins labeled with HRP (see above). To identify the spots corresponding to the proteins that bind BLf, the image obtained from the revealed membrane and the gel stained with Coomassie blue G-250 (Bio-safe, Bio-Rad) were merged, using image J software [36 ] by aligning MW markers and membrane borders. Obtaining OMP that bound BapoLf as well as overlay assays were performed in three independent experiments.
9
Isoelectric Focusing of Whole Cell Proteins
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The isoelectric focusing (IEF) was performed using the PROTEAN IEF Cell system (Bio-Rad). 500–600 μg of whole cell protein was added to 300 μl of rehydration buffer for 11 cm IPG strip (pH 4–7 non-linear, Bio-Rad). Protein sample was applied to the IPG strips and kept at room temperature (16–18 h) for passive rehydration. IEF was performed at 250 V for 20 min; 10,000 V for 2 h 50 min; 10,000–40,000 V/h and an optional step of 500–1000 V 10–15 h.
10
2D Gel Protein Standardization
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Five microliters of 2D gel protein standards (Bio-Rad, Hercules, CA, USA, #161-0320) were added to each sample tube. IPG strips (pH 3–10; 11 cm; Bio-Rad, #163-2014) were rehydrated overnight with these sample solutions. Iso-electric focusing was performed with a Protean IEF cell system (Bio-Rad, Hercules, CA, USA) using the standard protocol and a preset linear volt ramp program (8000 V and 50 μA/strip max., 35,000 vH). The focused strips were stored at −80 °C for later use.
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