Prior to printing of purified recombinant antigens, samples were diluted to 200 ng/μl in protein microarray printing buffer (50 mM HEPES, 140 mM NaCl, 2 mM DTT, pH 7.3) with glycerol (40%). Replicate spots (n = 6) of alphavirus proteins and controls were printed onto microporous-nitrocellulose-coated slides (ONCYTE SuperNOVA; Grace Bio-Labs, Inc., Bend, OR) with an Inkjet microarray printer (ArrayJet, Roslin, United Kingdom) at 65% humidity. Control proteins included bovine serum albumin (BSA), E. coli -expressed Rift Valley fever virus glycoprotein, and antigens from DENV and influenza virus hemagglutinin proteins (Immune Technology Corp., New York, NY), along with human, mouse, monkey, goat, and rabbit IgG (Rockland Immunochemicals Inc., Limerick, PA). The printed microarray slides were desiccated (12 h) and stored frozen (−20°C) until use. Spot deposition and the quality of spotted proteins were determined with SYPRO Ruby protein stain and mouse anti-polyhistidine monoclonal antibody (clone HIS-1; Sigma-Aldrich).
Sypro ruby protein stain
Manufactured by Merck Group
Sourced in Germany
SYPRO Ruby protein stain is a fluorescent stain used to detect and quantify proteins in polyacrylamide gels. It binds to a wide range of proteins and exhibits a high sensitivity, allowing the detection of nanogram quantities of protein.
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Lab products found in correlation
2 protocols using sypro ruby protein stain
1
Protein Microarray Printing Protocol
2
SDS-PAGE Protein Separation and Sypro Staining
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SDS-PAGE was run on a PhastGel gradient (10–15%) minigel system (GE Healthcare, Munich, Germany). For Sypro staining, proteins were fixed on the gel for 2 × 30 min in 50% MeOH and 7% acetic acid. The gel was then incubated with 2 ml Sypro Ruby protein stain (Sigma-Aldrich; Merck, Darmstadt, Germany) overnight at room temperature. After 30 min washing in 10% MeOH and 7% acetic acid and additional washing for 10 min in H2O, fluorescence signals were detected on a UVP ChemStudio imager (Analytik Jena, Jena, Germany).
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