Platelets from patients with myocardial infarction and non-ischemic chest pain were lysed in 100 µL of CHAPS lysis buffer (8M Urea, 4% CHAPS, 2% DTT) and purified using a 2D Clean-up kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions. Comparative 2D gel analysis of the proteomes was performed as described previously with slight modifications [34 (link)]. First dimension isoelectric focusing was performed using a Protean IEF Cell focusing unit (BioRad, Hercules, CA, USA) with pH 3–10NL gel strips (11 cm, GE Healthcare, for some subsequent gels pH 4–7). For the second dimension, equilibrated (5.7M Dithiothreitol and 1.5M Iodoacetamide) gel strips were applied to 12% polyacrylamide gels. Proteins were stained with Lava purple fluorescent staining (Fluorotechnics, Sydney, Australia). Protein spots of interest were excised manually from the gels, digested with trypsin and analyzed by LC ESI-MS/MS as described previously [35 (link)]. Peptide sequence analysis by mass spectrometry of the isolated protein spots resulted in the identification of the differential cleavage of kindlin-3 in the myocardial infarction group.
Protean ief cell focusing unit
Manufactured by Bio-Rad
Sourced in United States
The Protean IEF Cell focusing unit is a laboratory equipment designed for isoelectric focusing (IEF) of proteins. It provides a controlled environment for the separation and analysis of proteins based on their isoelectric point. The device features a temperature-controlled chamber and supports the use of various IEF gel formats.
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Lab products found in correlation
3 protocols using protean ief cell focusing unit
1
Differential Proteomics of Platelets in Myocardial Infarction
2
Proteomic Profiling of Platelets
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Platelets from patients with myocardial infarction and nonischemic chest pain were lysed in 100 µL of CHAPS lysis buffer (8 M Urea, 4% CHAPS, and 2% DTT) and purified using a 2D clean-up kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions. Comparative 2D gel analysis of the proteomes was performed as described previously with slight modifications [34 (link),35 (link)]. First dimension isoelectric focusing was performed using a Protean IEF Cell focusing unit (BioRad, Hercules, CA, USA) with pH 3–10 NL gel strips (11 cm, GE Healthcare, for some subsequent gels pH 4–7). For the second dimension equilibrated (5.7 M Dithiotreitol and 1.5 M Iodoacetamide) gel strips were applied to 12% polyacrylamide gels. Proteins were stained with Lava purple fluorescent staining (Fluorotechnics, Sydney, Australia). Protein spots of interest were excised manually from the gels, digested with trypsin. and analyzed by LC ESI-MS/MS (Applied Biosystems/MDS Sciex, Darmstadt, Germany) as described previously [35 (link)].
3
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.
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