Qc colloidal coomassie stain

Binding affinities between FH19-20 or FH (from Complement Technologies, US) and wild type and mutants of FhbA proteins was determined using Microscale Thermophoresis (MST) with a Monolith NT.115 instrument (Nanotemper Technologies, Germany). FH19-20 and FH were labelled with RED-tris-NTA dye (Nanotemper Technologies, US) in PBS according to the manufacturer’s instructions. 10 μl of 300 nM labelled protein was mixed with 10 μl of ligand in PBS/0.025% Tween-20, the mixture loaded into standard treated capillaries (Nanotemper Technologies, US) and thermophoresis was measured at 22° C for 22–30 s with 20% LED power and 20%/60% infrared laser power. Three independent measurements were made, and results were analysed using the MO. Affinity Analysis software version 2.1 (Nanotemper Technologies, US). For gel filtration 100 μl of proteins (20 nmole) were eluted individually and in combination with FH19-20 on a Superdex 200 Increase 10/300 GL column attached to an ÄKTA (GE-healthcare) with PBS buffer at +4° C. 1 ml fractions from each run were collected and subjected to SDS-PAGE analyses on TGX gradient (4–20%) precast mini-gels (Biorad, CA, US), which were fixed, stained with QC Colloidal Coomassie Stain (Biorad), and proteins were visualized using Image Lab (Biorad). For high salt experiments, PBS buffer supplemented with 500 mM NaCl was used.

The construction of DENVLP1-4 expression plasmids and production of VLPs has been reported previously (22 (link)). Briefly, Freestyle 293 F cells (Thermo Fisher Scientific) cultured in FreeStyle 293 Expression Medium (Thermo Fisher Scientific) were transfected with DENV VLP-expressing plasmids. The culture supernatant at 4 days after transfection was clarified, concentrated, and purified by HiTrap Q XL (GE Healthcare Life Sciences) and Foresight CHT Type II (Bio-Rad) columns with sodium phosphate gradient. Total protein concentration of purified DENV VLP was measured by Quick Start Bradford Protein Assay (Bio-Rad) with a final yield of up to 10 mg/L for all four serotypes. Purity of the DENV VLP was assessed by SDS-PAGE followed by Coomassie dye-based staining using QC colloidal Coomassie stain (Bio-Rad). The morphologies of DENV VLPs were analyzed at the National Institute of Infectious Disease in Japan (NIID) Microscope Facility to confirm consistency prior to their use in animal studies.

This analysis was carried out via vertical polyacrylamide gel electrophoresis under denaturing and reducing conditions (SDS-PAGE) using a Mini-protean II system (Bio-Rad, South Granville, NSW, Australia). Each sample was mixed with an equal volume of 2X Laemmli loading buffer (100 mM Tris–HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, and 20% glycerol) (Bio-Rad, Hercules, CA, USA). Samples were loaded onto a 4–20% gradient TGX™ polyacrylamide gel (Bio-Rad, Hercules, CA, USA) along with a Precision Plus™ Protein molecular mass marker (Bio-Rad, Hercules, CA, USA). Proteins were visualized with QC Colloidal Coomassie Stain (Bio-Rad, Hercules, CA, USA). Molecular mass calculation was performed based on the molecular mass marker using the ImageLab 4.1 point-to-point method [32 ].

Folded oligonucleotides (0.15 nmols) were separated on a 20% polyacrylamide gel in 0.5× TBE/0.5× IB1 (45 mM Tris-borate, 1 mM EDTA, 50 mM KCl, 25 mM NaCl, 10 mM NaH₂PO₄/ Na₂HPO₄, pH 8, or pH 7 for i-motifs) at 4°C. The gel was rinsed with H₂O, stained with 0.35% Stains-All (Merck) in 50% formamide for 15 min, de-stained in H₂O, and then imaged.
For the analysis of complex formation, purified his-tagged HP1 proteins (0.2 nmols) mixed with 0.3 nmols TERRA45 in 1× IB1 were separated on a 5% polyacrylamide gel in 0.5× TBE/0.5× IB1 at 4°C. The gel was then stained with QC Colloidal Coomassie Stain (Bio-Rad, USA) for 18 h at room temperature, de-stained with H₂O and imaged.

Analysis of protein pattern were performed as previously seen^{53 (link)}. Total protein concentrations in extracts were determined by the Bradford method with bovine serum albumin (BSA) as the standard. Then, SDS-PAGE gel electrophoresis was performed on samples with equalized concentrations of total protein. In Particular 20 μl of samples was mixed with 20 μl of Laemmli Sample Buffer with β-mercaptoethanol (Bio-Rad) and heating (95 °C, 5 min). then 40 μl of mixed samples and 5 μl of protein marker (Precision Plus Protein Dual Color Standards, Bio-Rad) were loaded onto the gel and resolved, using the Mini-PROTEAN electrophoresis system (Bio-Rad). The protein bands, separated on gel, were fixed, stained in QC Colloidal Coomassie Stain and destained solution according to the producer procedure (Bio-Rad).