Samples were loaded on a 4–15% PROTEAN TGX Stain-Free Protein Gel (Bio-Rad, 4568083 (Mini) or 5678084 (Midi)) with Tris/Glycine/SDS running buffer (Bio-Rad, 1610772), and proteins were resolved at a current of 125 V (Mini) or 150 V (Midi) for 1 h at room temperature. In-gel fluorescence and stain-free total protein signal were detected using a Bio-Rad Chemidoc MP Imaging System. Band intensities were quantified using Bio-Rad Image Lab Software v6.0.
Protean tgx stain free protein gel
Manufactured by Bio-Rad
Sourced in France, Switzerland
The PROTEAN TGX Stain-Free Protein Gel is a precast polyacrylamide gel used for the separation and visualization of proteins. It is designed to enable fast and efficient protein electrophoresis without the need for traditional staining methods.
Automatically generated - may contain errors
Lab products found in correlation
Tris glycine sds running buffer, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Lab software v6, by Bio-Rad (2 mentions)
Kta pure chromatography system, by GE Healthcare (1 mentions)
Hitrap, by Cytiva (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Laemmli buffer, by Merck Group (1 mentions)
Sodium orthovanadate, by Merck Group (1 mentions)
Tris glycine sodium dodecyl sulfate buffer, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
4 protocols using protean tgx stain free protein gel
1
Stain-Free Protein Gel Electrophoresis
2
Stain-Free Protein Gel Electrophoresis
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Samples were loaded on a 4–15% PROTEAN TGX Stain-Free Protein Gel (Bio-Rad, 4568083 (Mini) or 5678084 (Midi)) with Tris/Glycine/SDS running buffer (Bio-Rad, 1610772), and proteins were resolved at a current of 125 V (Mini) or 150 V (Midi) for 1 h at room temperature. In-gel fluorescence and stain-free total protein signal were detected using a Bio-Rad Chemidoc MP Imaging System. Band intensities were quantified using Bio-Rad Image Lab Software v6.0.
3
Recombinant Expression and Purification of CPL Proteins
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The recombinant N-terminally His6-MBP-tagged CPL proteins were expressed in BL21 (DE3) pLysS (Novagen, Watertown, MA 02472 USA) E. coli cells upon selection with 100 µg mL−1 ampicillin. Cultures (50 mL or 300 mL) were grown for 40 h at 25 °C in ZYM5052 auto-inducing media. Upon bacterial lysis the proteins were purified on a MBP Trap High Performance (HP) column (GE Healthcare Life Science, Freiburg, Germany) in an ÄKTA Pure chromatography system (GE Healthcare Life Science, Freiburg, Germany) and eluted with 10 mM maltose in phosphate-buffered saline (PBS). Peak fractions were pooled and dialyzed against PBS pH 7.4 using a HiTrap Desalting (DST) column (GE Healthcare Life Science, Freiburg, Germany). Protein concentrations were determined with NanoDrop 2000 UV-Vis equipment (Thermo Scientific, Wilmington, DE, USA). Protein expression and purification steps were monitored by analysis of total, soluble and eluted fractions by SDS-PAGE using pre-casted polyacrylamide gels (PROTEAN TGX Stain-free protein gels, Biorad, Marnes-la-Coquette, France).
4
Sema3A Signaling Pathway Analysis
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Before protein extraction, cells were treated with CPL-K, CPL-F, or CPL at 10−6 M for 1 h and then stimulated with Sema3A at 100 ng/mL for 30 min as previously described elsewhere [44 (link)]. After lysis in Laemmli buffer (Sigma) supplemented with protease inhibitors (Roche, Basel, Switzerland) and phosphatase inhibitor (sodium orthovanadate, Sigma-Aldrich), protein samples were separated in 4–20% pre-casted polyacrylamide gels (PROTEAN TGX Stain-free protein gels, Biorad) by SDS gel electrophoresis in Tris/Glycine/Sodium Dodecyl Sulfate buffer (Biorad) at 300 V for 18 min. Proteins were transferred onto nitrocellulose membrane using the Trans-Blot® TurboTM Transfer System (Trans-blot Turbo, Biorad) and antibodies Akt and phospho-Akt (Cell signaling), and their respective secondary antibodies coupled with HRP (Biorad) were used. The blots were developed with ECL (Biorad), imaged with a bio-imager (ChemidocTM Touch Imaging System, Biorad), and normalized using the stain-free technology (western blot figures can be found in the Supplementary Figure S1 ).
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