Hybond pvdf membrane

The Western blotting protocol was described in a previous study [34 (link)]. Briefly, cells were harvested, washed twice with PBS, and lysed in ice-cold RIPA lysis buffer. Lysates were boiled in 1× SDS-sample buffer and resolved through SDS-PAGE. SDS-PAGE-separated proteins were transferred through electrophoresis onto a Hybond-PVDF membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Finally, blots were probed with the reported primary antibodies and appropriate secondary antibodies.

The analysis of the expression of the fluorescent Citrine protein linked to different N-terminal tags was done as previously described [3] (link). Bacterial cell aliquots of 1 ml of culture were harvested at mid-exponential growth phase. Cells were incubated at 37°C during 30 minutes in deoxicholate (0.25 mg/ml), RNase (10 mg/ml), DNase (10 mg/ml) and PMSF (1 mM). For the fluorescent protein analysis, proteins were incubated with solubilization buffer (200 mM Tris-HCl pH 8.8, 20% glycerol, 5 mM EDTA pH 8.0, 0.02% bromophenol blue, 4% SDS, 0.05 M DDT) at 37°C during 5 minutes and separated on SDS-PAGE. Gel images were acquired on a FUJI FLA 5100 laser scanner (Fuji Photo Film Co.) with 635 nm excitation and >665 nm band pass emission filter for protein molecular weight marker detection and 473 nm excitation and >510 nm band pass emission filter for Citrine detection.
For western-blot analysis, cells extracts were boiled during 5 minutes before being separated on SDS-PAGE. Proteins were transferred into a Hybond PVDF Membrane (Amersham) and probed with Living Colors Av. Peptide Antibody (Clontech) for the detection of Citrine, used at 1∶500, followed by 1∶100000 of goat anti-rabbit conjugated to horseradish peroxidase. Detection was done with ECL Plus Western Blotting Detection Reagents (Amersham).

Recombinant NS1 (final concentration at 0.03 μg/μL for DENV-1, -3, and -4, and 0.01 μg/μL for DENV-2) were prepared in PBS and 4X Laemmli Sample Buffer (Bio-Rad). The samples were divided into 2 tubes: (1) untreated and (2) incubated at 90°C for 10 min. The proteins were separated on an Any kD™ Mini-PROTEAN® TGX Stain-Free™ Protein Gels (Bio-Rad) and then transferred onto a Hybond PVDF membrane (0.45μm, Amersham). The membrane was incubated in PBS supplemented with 5% (w/v) skim milk overnight at 4°C, then purified antibodies at 1μg/mL for 2 h at RT. The membranes were washed 3 times with PBST, 5 minutes each, then peroxidase-conjugated anti-human IgG (1/10,000 dilution, Sigma Aldrich) was added for 1 h at RT. The membranes were washed three times with PBST, chemiluminescent substrates (WesternBright Sirius chemiluminescent Detection kit, Advansta) were added and then developed using BioRad ChemiDoc system. For SDS-PAGE, the gel was stained with InstantBlue^TM (Expedeon).