Similar gels were used for IEF and NEPHGE. Gels consisted of 4.5% acrylamide, 2% ampholytes pH 3–10 (Serva, Heidelberg, Germany), 4 M urea and 2% CHAPS. Gels were always prepared maximum 24 h before use. Minimum polymerization time was 1.5 h at 34°C. Polymerization was triggered by 0.1% ammoniumpersulfat (APS) and 0.01% N,N,N′,N′-tetramethylethylendiamin (TEMED). Sample buffer was prepared as 4× buffers for both separation methods (IEF, NEPHGE). Samples loaded on the gel contained 1 M urea, 10% glycerol, 0.5% CHAPS and 2% ampholytes. Before samples were applied, a pre-run of the gels was accomplished for 45 min at 30 V with no further restrictions. Electrophoresis conditions were described by Lüthje et al. (2014 ). For NEPHGE, the polarity and the IEF buffer system was reversed (Figure 1 ). Electrophoresis conditions for native NEPHGE were constantly set to 450 Vh, to keep highly alkaline proteins in the gel (step 1: 100 V, 150 Vh; step 2: 250 V, 250 Vh; step 3: 500 V, 200 Vh).
Ampholyte
Manufactured by Serva Electrophoresis
Sourced in Germany
Ampholytes are chemical compounds used in isoelectric focusing, a technique in electrophoresis. They establish a stable pH gradient within a gel or solution, which is essential for the separation and analysis of proteins and other biomolecules based on their isoelectric points.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Helicon (2 mentions)
Dithiothreitol (dtt), by Bio-Rad (1 mentions)
Urea, by Bio-Rad (1 mentions)
Mini ief cell model 111, by Bio-Rad (1 mentions)
Sypro ruby, by Lonza (1 mentions)
Protean plus dodeca cell, by Bio-Rad (1 mentions)
Protean ief cell focusing unit, by Bio-Rad (1 mentions)
Servalyte 3 10, by Serva Electrophoresis (1 mentions)
Acetic acid, by ITW Reagents (1 mentions)
Coomassie brilliant blue g 250, by ITW Reagents (1 mentions)
6 protocols using ampholyte
1
Isoelectric Focusing for Proteomic Analysis
2
Extraction of Epithelial-Mesenchymal Transition Proteins
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For the determination of EMT-associated proteins by two-dimensional electrophoresis (2-DE) whole cellular proteins were extracted from confluent and sparsely growing SW480 and HCT116 cells [22] (link). Protein samples were mixed with urea (9M), DTT (70 mM) (both BioRad, Munich, Germany) and Ampholytes (2%, Servalytes pH 5–7, Serva, Heidelberg, Germany), cleared by centrifugation (14,000 rpm/10 min/4°C) and frozen in liquid nitrogen. For standard polyacrylamide gel electrophoresis (PAGE) proteins were extracted with triple-detergent lysis buffer [23] supplemented with a protease inhibitor cocktail [22] (link), cleared by centrifugation and finally frozen in liquid nitrogen.
3
Isoelectric Focusing of Rat Urine Proteins
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Isoelectric focusing (IEF) was performed using a Model 111 Mini IEF Cell (Bio-Rad, Hercules, CA, USA). The rat urine samples were de-salted, freeze-dried, dissolved in deionized water, and electrophoresed in a 5% polyacrylamide gel containing 5% ampholytes (pH range of 3–7, SERVA Electrophoresis GmbH, Germany). The gels were fixed, stained, destained, and then imaged with a ChemiDoc MP system (Guo et al. 2018 (link)).
4
Proteomic Analysis of TPCN1 KO Mice
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Isoelectric focusing (IEF) was performed in pH 4-7 immobilized pH gradient (IPG) strips of 24 cm (GE Healthcare, UK). 500 μg of protein from LV of TPCN1 KO and WT mice independently was applied to each strip, and subjected to passive rehydration in 250 μL of 2-DE extraction buffer supplemented with ampholytes (0.1% servalyte 3-10, 0.05% servalyte 9-10 (SERVA, DE)) during 24 h after centrifugation at 13000g for 5 min. Passive rehydration was followed by active rehydration at 50 V during 12 h before IEF, performed in a Protean IEF Cell focusing unit (BioRad, USA) until 20000 V/total h were reached.
Subsequently to IEF, the IPG strips were equilibrated for 15 min in 4 M urea, 2 M thiourea, 50 mM Tris pH 6.8, 2% sodium dodecyl sulphate (SDS), 12 mM dithiothreitol (DTT) and 30% glycerol. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12% polyacrylamide gels using a Protean Plus Dodeca Cell (BioRad, USA) at 15 mA/ gel for 12 h or until the dye front reached the bottom of the gel, at 18°C constant temperature.
The 2-DE gels were stained with Sypro Ruby (Lonza, CH) following manufacturer's instructions.
Subsequently to IEF, the IPG strips were equilibrated for 15 min in 4 M urea, 2 M thiourea, 50 mM Tris pH 6.8, 2% sodium dodecyl sulphate (SDS), 12 mM dithiothreitol (DTT) and 30% glycerol. SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12% polyacrylamide gels using a Protean Plus Dodeca Cell (BioRad, USA) at 15 mA/ gel for 12 h or until the dye front reached the bottom of the gel, at 18°C constant temperature.
The 2-DE gels were stained with Sypro Ruby (Lonza, CH) following manufacturer's instructions.
5
Proteomic Analysis of Porcine Muscle
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Chemical reagent: Urea, Thiourea, Ditiothreitol, Sodium hydroxide (NaOH), Glycerol, Sodium dodecyl sulfate (SDS), Tris, Acrylamide, Ammonium persulfate (APS), 2-Propanol, Acetic acid (PanReac, Spain); Bis-acrylamide, Tetramethylethylenediamine (TEMED), Mercaptoethanol, Bromophenol blue, Glycine, Coomassie Brilliant Blue G-250, Triton X-100 (Helicon Russia); Ampholyte (Serva, Germany) and Phosphoric (V) acid (H3PO4) (Componentreaktiv, Russia) .
The object of the study was the Vietnamese Pot-bellied from healthy females of 60 -65 days old pig L. dorsi muscle. Samples were taken within 20 minutes after slaughter and placed in dry ice. Frozen muscle tissues (50 mg) were homogenized in 1 mL 7 M Urea, 2 M Thiourea, 1% Ditiothreitol, 0.4% Triton X-100, 2% pH 3-10 Ampholyte. Homogenates were centrifugated with an acceleration of 20 000 g for 20 minutes. Three samples, obtained from different animals, were studied by twodimensional electrophoresis in two variations of isoelectric focusing (IEF) and two methods of gel incubation after IEF. Figure 1 shows the experimental design.
The object of the study was the Vietnamese Pot-bellied from healthy females of 60 -65 days old pig L. dorsi muscle. Samples were taken within 20 minutes after slaughter and placed in dry ice. Frozen muscle tissues (50 mg) were homogenized in 1 mL 7 M Urea, 2 M Thiourea, 1% Ditiothreitol, 0.4% Triton X-100, 2% pH 3-10 Ampholyte. Homogenates were centrifugated with an acceleration of 20 000 g for 20 minutes. Three samples, obtained from different animals, were studied by twodimensional electrophoresis in two variations of isoelectric focusing (IEF) and two methods of gel incubation after IEF. Figure 1 shows the experimental design.
6
Proteomic Analysis of Muscle Tissue
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Chemical reagents: Urea, Thiourea, Ditiothreitol, Sodium hydroxide (NaOH), Glycerol, Sodium dodecyl sulfate (SDS), Tris, Acrylamide, Ammonium persulfate (APS), 2-Propanol, Acetic acid, Bis-acrylamide, Tetramethylethylenediamine (TEMED), Mercaptoethanol, Bromophenol blue, Glycine, Coomassie Brilliant Blue G-250 (PanReac, Spain); Triton X-100 (Helicon Russia); Ampholyte (Serva, Germany) and Phosphoric (V) acid (H3PO4) (Component-reaktiv, Russia).
Samples were taken within 20 minutes after slaughter and placed in dry ice. Frozen muscle tissues (50 mg) were homogenized in 1 mL 7 M Urea, 2 M Thiourea, 1 % Ditiothreitol, 0,4 % Triton X-100, 2% pH 3 -10 Ampholyte. Homogenates were centrifugated with an acceleration of 20 000 g for 20 minutes. Three samples, obtained from different animals, were studied by twodimensional electrophoresis in three replicates.
Samples were taken within 20 minutes after slaughter and placed in dry ice. Frozen muscle tissues (50 mg) were homogenized in 1 mL 7 M Urea, 2 M Thiourea, 1 % Ditiothreitol, 0,4 % Triton X-100, 2% pH 3 -10 Ampholyte. Homogenates were centrifugated with an acceleration of 20 000 g for 20 minutes. Three samples, obtained from different animals, were studied by twodimensional electrophoresis in three replicates.
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