Isoelectric focusing (IEF) was carried out as previously described [11 ], using BioRad PROTEAN® IEF Cell and 7-cm, pH 3–10 nonlinear Immobilized pH Gradient (IPG) strips (BioRad). Unless indicated otherwise in Figure Legends, heat treatments of cell extracts and reconstituted protein–NA mixtures were omitted. The second (SDS-PAGE) stage was carried out using Mini-PROTEAN® II Cell (BioRad) largely according to the manufacturer’s manual, except that custom-made Teflon combs with a side mini-well for Precision Plus Dual Color Standards (BioRad) were used. Following the IEF stage, the 2D-fractionated native proteins and protein–NA complexes were transferred onto Immobilon®-P membranes (Millipore), typically, for 48–60 hours at 80 V (in a cold room), in 1x Tris Glycine Transfer Buffer, pH 8.3 (TGTB) supplemented with 10% HPLC-grade methanol. TGTB was obtained as a 10x stock from KD Medical. Immunostaining of the membranes with Hfq-specific antibodies [9 ] and chemiluminescent detection and quantitation of the protein were carried out as previously described [9 ]. Each 2D-PAGE/IB experiment was repeated at least twice, with varied modes of sample pre-treatment (as seen in Figs. 2A and 2B ).
Biorad protean ief cell
Manufactured by Bio-Rad
Sourced in Sweden
The BioRad Protean IEF Cell is a laboratory equipment used for isoelectric focusing (IEF), a technique in protein separation and analysis. It provides a controlled environment for running IEF experiments, allowing for the separation of proteins based on their unique isoelectric points.
Automatically generated - may contain errors
Lab products found in correlation
Precision plus dual color standard, by Bio-Rad (1 mentions)
Mini protean 2 cell, by Bio-Rad (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Agarose, by Avantor (1 mentions)
Glycerol, by Kanto Chemical (1 mentions)
Iodoacetamide, by Avantor (1 mentions)
Sodium dodecyl sulfate (sds), by Avantor (1 mentions)
3 protocols using biorad protean ief cell
1
Two-Dimensional Protein-Nucleic Acid Complex Analysis
2
Two-Dimensional Protein Separation Protocol
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Extracted protein (proximally 500 µg) was added to thiourea rehydration buffer (7 M urea, 2 M thiourea 2 CHAPS (w/v), 1% bromophenol blue, and 2% (v/v) IPG buffer). The mixture was applied on 18-cm IPG gel strips (pH 3–10 nonlinear (NL)) (GE Healthcare Bio-Sciences AB, USA) and incubated at room temperature for 45 min. Then, actively rehydrated for 16 h at 50 V. Strips were then focused for 60000Vh at 20ºC within the BioRad Protean IEF Cell to separate proteins according to their isoelectric point (PI).
After IEF, the strips were equilibrated and reduced in the presence of equilibration buffer 6 M urea, 29.3% (v/v) glycerol, 2% (w/v) SDS, and 75 mM Tris-HCL containing 2% (w/v) DTT and then in the equilibration buffer containing 5% (w/v) iodoacetamide.
The equilibrated IPG strips were loaded onto the top of a 12% (w/v) SDS-PAGE and the proteins were separated according to their molecular weight (MW) using BioRad Protean IIxi. Then, the gels were visualized using a modified Coomassie brilliant blue G-250 (CBB) staining method.
After IEF, the strips were equilibrated and reduced in the presence of equilibration buffer 6 M urea, 29.3% (v/v) glycerol, 2% (w/v) SDS, and 75 mM Tris-HCL containing 2% (w/v) DTT and then in the equilibration buffer containing 5% (w/v) iodoacetamide.
The equilibrated IPG strips were loaded onto the top of a 12% (w/v) SDS-PAGE and the proteins were separated according to their molecular weight (MW) using BioRad Protean IIxi. Then, the gels were visualized using a modified Coomassie brilliant blue G-250 (CBB) staining method.
3
Protein Separation and Characterization via IEF and SDS-PAGE
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Isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were conducted as described previously [45 (link)]. The 18-cm immobibline dry strips (pH 4–7) (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were rehydrated within the BioRad Protean IEF Cell for 16 h at 20 °C with 300 μL rehydration buffer including 200 μg protein lysates respectively prepared from Sh-Gal-1(+120) and Sc-Gal-1(+120) T24 cells. Then, the proteins were concentrated at 20 °C at 50, 100, 200, 500, 1000, 5000, and 8000 V, respectively, with a total of 81,434 voltage-hours. After isoelectric focusing, the gel strips were equilibrated in the equilibration buffer (6 M urea, 30% (v/v) glycerol (Kanto Chemical, Portland, OR, USA), 2% (w/v) SDS (aMResco, Solon, OH, USA)) containing 2% (w/v) DTT for 15 min and then in the equilibration buffer containing 5% (w/v) iodoacetamide (aMResco, Solon, OH, USA) for a further 15 min. The equilibrated gel was loaded onto the top of a 12.5% (w/v) polyacrylamide gel and sealed with 0.5% (w/v) agarose (aMResco, Solon, OH, USA) and the proteins were separated at 420 V using BioRad Protean IIxi until bromophenol blue reached the bottom of the gel.
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