Pageruler unstained protein ladder

A standard PURE reaction programmed with 17.5 nM (250 ng) of a linear Rep DNA template (under control of a T7 promoter) was supplemented with 0.6 µl FluoroTect™ Green_Lys tRNA (FluoroTect™ Green_Lysin vitro translation labelling system, Promega). Template DNA was omitted in the negative control reaction. Samples were incubated for 2  h at 37 °C in a nuclease-free PCR tube (Thermo Fisher Scientific) using a ProFlex PCR System (Thermo Fisher Scientific) and subsequently treated with 0.6 μl RNase Cocktail™ Enzyme Mix (0.5 U/μl RNase A and 20 U/μl RNase T1, Thermo Fisher Scientific) for 15 min at 37°C to degrade non-incorporated Green_Lys tRNA. 7.5 μl sample were mixed with an equal volume 2× Laemmli sample loading buffer (incl. 200 mM DTT) and denatured for 2.5 min at 65°C. Samples were analysed by conventional discontinuous SDS-PAGE (10% gel) run at 4°C (100 V for 10 min, then 200 V) on a Midi-format electrophoresis system (Atto). The fluorescent signal of the de novo expressed rep β subunit was imaged on a fluorescence laser scanner (Typhoon FLA 9000, GE Healthcare) at either 473 nm (blue LD laser/510LP filter) or at 532 nm (green SHG laser/575LP filter). Total protein and the molecular-weight size marker (PageRuler™ Unstained Protein Ladder, Thermo Fisher Scientific) were visualized after SYPRO Ruby (Bio-Rad) staining using the same instrument (473 nm, blue LD laser/575LP filter).

Purified PA-SR01 was diluted in SDS buffer (5:1, vol/vol) and heated at 95°C for 5 min. The sample was then resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using a Mini-PROTEAN Tetra Cell (Bio-Rad Laboratories). The Mini-PROTEAN TGX stain-free precast gel was run in an SDS running buffer (pH 8.3) at 120 V for 1.5 h using a PageRuler unstained protein ladder (Thermo Fisher) for size calibration.

Protein concentrations were quantified using Bradford protein assay (Carl Roth, Germany) following TCA/acetone precipitation. Bovine serum albumin (BSA) was chosen for modeling standard curves and measurements were recorded in triplicates in 96-well plates on EnSpire® Multimode Plate Reader (PerkinElmer, USA). Protein extracts were conveyed on 12% one-dimensional SDS polyacrylamide gel electrophoresis, using Bio-Rad Mini-Protean II Equipment and PageRuler ™ Unstained Protein Ladder (ThermoFischer Scientific, USA), to assess the gross qualitative variances in protein profiles. After electrophoresis, gels were stained with Coomassie brilliant blue (CBB) R-250.

Recombinant proteins (TRX-h3, -h5, -f1, -m4 and GRX-C2) were prepared and treated with OPDA as described before. After 1 h of incubation, mPEGmal-5000 was added to 2 µg of protein to a final concentration of 1 mM. Samples were incubated at RT and 300 rpm for 1 h and directly mixed with 5× loading dye (225 mM Tris pH 6.8, 5% (w/v) SDS, 0.05% (w/v) bromophenol blue, 50% (v/v) glycerol, 200 mM DTT) and heated at 95 °C for 5 min.
Analysis of samples was performed by SDS-PAGE (separation at 50 mA for 80 min) and subsequent Coomassie brilliant blue staining. Pageruler Unstained Protein Ladder (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was used as molecular mass standard.

Protein profiles were obtained by combining 25 μl of culture supernatant supplemented with 5 μl of 5 × Laemmli Loading Buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 0.1 M dithiothreitol, 0.2 mg/ml Bromophenol Blue) and separating this on 12% SDS-PAGE gels. Proteins were visualized by silver staining and a PageRuler Unstained Protein Ladder (Thermo Scientific) was used as protein marker.

For electrophoresis analysis, the recovered proteins were diluted with PBS to adjust the sample buffer, and 40 µl of the proteins were separated on a 12% SDS-polyacrylamide gel (Bolt™ 4–12% Bis-Tris Plus Gels, 10-well) at the constant voltage of 160 V. A PageRuler Unstained Protein Ladder (Thermo Scientific) as the molecular weight standard (10–200 kDa) was also run on the gels. For CY5-labeled samples, the gels were first imaged by an Amersham Typhoon NIR laser scanner. Then, the protein bands were detected by Imperial Protein stain (Coomassie brilliant blue, Thermoscientific). The stained gels were scanned on a Bio-Rad gel documentation system. Protein quantification was performed using the plot profile tool in Fiji/ImageJ (ref). The staining intensity and run length were normalized based on the maximum values.