Fractions from size exclusion were boiled for 5 minutes in loading buffer (final concentrations of 60 mM Tris-HCl pH 6.8, 2% SDS, 10% v/v glycerol and 0.01% bromophenol blue) and run on Mini-Protean 4-15% TGX-SDS-PAGE in llaemli running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS pH 8.3) at 300 V for 20 minutes followed by staining with InstantBlue (Expedeon). Molecular weight ladder used is PageRuler Unstained (ThermoFisher). Samples for native PAGE were mixed 3:1 with sample buffer (1x TBE, 20% v/v glycerol, 0.04% Bromophenol blue) and run at 90 V for 2h on a 4.5% native PAGE in 0.5x TBE buffer (1x TBE is 90 mM Tris-borate pH 8.3; 1mM EDTA).
Pageruler unstained
Manufactured by Thermo Fisher Scientific
Sourced in Canada
The PageRuler Unstained is a pre-stained protein ladder used for estimating the molecular weight of proteins in SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) analysis. It contains a set of unstained protein bands with known molecular weights, providing a reference for comparison during protein separation and identification.
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5 protocols using pageruler unstained
1
SDS-PAGE and Native PAGE Analysis
2
Visualizing Radiolabeled Cell-Free Toxins
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using precast gels (NuPAGE, 10% Bis-Tris, Life technologies) was performed to confirm the molecular mass of radio-labeled cell-free synthesized toxins. Therefore, 3 µl aliquots of the toxin fraction were precipitated in cold acetone (Carl Roth GmbH) as described previously 69 (link) . Protein samples were dissolved in LDS samples buffer (NuPAGE, Life technologies) and SDS-PAGE was performed under non-reducing and reducing (addition of 50 mM DTT, Aplichem GmbH) conditions. SeeBlue™ Plus2 protein standard and Page Ruler Unstained (Thermo Fisher Scientific Inc.) were used as a protein size determination. Proteins were separated on precast NuPAGE SDS-PAGE gels for 35 min at 185 V and were stained with a self-prepared Coomassie Brilliant Blue G250 solution (Serva, 75 mg/L and 35 mM HCL, Carl Roth GmbH). Subsequently, gels were dried on Whatman paper for 70 min at 70 °C (Unigeldryer 3545D, Uniequip) and dried gels were exposed to phosphor screens (GE Healthcare) for a minimum of three days. The protein standard bands were marked using 14 (link) C-Leucin ink to be detectable on the autoradiographs. Finally, radio-labeled proteins were visualized using a phosphor imager system (Amersham RGB Imager, GE Healthcare).
3
SDS-PAGE and Native PAGE Analysis
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Fractions from size exclusion were boiled for 5 minutes in loading buffer (final concentrations of 60 mM Tris-HCl pH 6.8, 2% SDS, 10% v/v glycerol and 0.01% bromophenol blue) and run on Mini-Protean 4-15% TGX-SDS-PAGE in llaemli running buffer (25 mM Tris, 192 mM Glycine, 0.1% SDS pH 8.3) at 300 V for 20 minutes followed by staining with InstantBlue (Expedeon). Molecular weight ladder used is PageRuler Unstained (ThermoFisher). Samples for native PAGE were mixed 3:1 with sample buffer (1x TBE, 20% v/v glycerol, 0.04% Bromophenol blue) and run at 90 V for 2h on a 4.5% native PAGE in 0.5x TBE buffer (1x TBE is 90 mM Tris-borate pH 8.3; 1mM EDTA).
4
Outer Membrane Protein Analysis
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Outer membrane isolates were diluted (1:1.5) with sample buffer (100 mm Tris/HCl (pH 6.8) containing 4% SDS, 0.2% bromophenol blue, 200 mm dithiothreitol and 20% glycerol), boiled for 10 minutes and analyzed on 10% polyacrylamid gels. Proteins were stained with Coomassie brilliant blue (R250). To correlate molecular masses of protein bands of interest, a molecular weight standard was used (PageRuler unstained, Fermentas, Burlington, Canada).
5
Recombinant Protein Expression in E. coli
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WT CXCL12 was chemically synthetized by F. Baleux, Pasteur Institute, Paris and the WT CCL5 was purchased from Immunotools. The molecular weight markers (PageRuler Unstained and PreStained Protein Ladder) were purchased from Fermentas.
E. coli BL21 (DE3) cells transformed with the different expression plasmids were grown at 37°C in Luria Bertani medium supplemented with 100 µg.mL−1 ampicillin until cultures reached an optical density of 0.6 to 0.8 at 600 nm. Then, 1 mM isopropylthio-β-D-galactopyranoside was added to the cultures in order to induce expression of recombinant proteins. Cells were further incubated for 3 h at 37°C (CXCL12-HIS, CXCL12-Strep and CCL5-Strep) or 16 h at 20°C (MBP-CXCL12-Strep, MBP-CCL5-Strep, CXCL12-LT-HIS, CXCL12-LT-Strep and CCL5-LT-Strep) and harvested by centrifugation for 30 min at 5,000 g.
E. coli BL21 (DE3) cells transformed with the different expression plasmids were grown at 37°C in Luria Bertani medium supplemented with 100 µg.mL−1 ampicillin until cultures reached an optical density of 0.6 to 0.8 at 600 nm. Then, 1 mM isopropylthio-β-D-galactopyranoside was added to the cultures in order to induce expression of recombinant proteins. Cells were further incubated for 3 h at 37°C (CXCL12-HIS, CXCL12-Strep and CCL5-Strep) or 16 h at 20°C (MBP-CXCL12-Strep, MBP-CCL5-Strep, CXCL12-LT-HIS, CXCL12-LT-Strep and CCL5-LT-Strep) and harvested by centrifugation for 30 min at 5,000 g.
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