Sephadex columns

TentaGel HL-COOH beads were obtained from Rapp Polymere (www.rapp-polymere.com). Ovalbumin (OVA), fluorescein isothiocyanate (FITC), N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), dimethylformamide (DMF), 3,3’,5,5’-tetramethylbenzidine (TMB) were purchased from Sigma-Aldrich (www.sigmaaldrich.com). N,N'-disuccinimidyl carbonate (DSC) was acquired from Fluka (Buchs, Switzerland). The anti-CBZ IgG (ordering code BAM-mab 01 (CBZ)) and CBZ-HRP were produced as described elsewhere [25 ]. Anti-mouse IgG-Alexa647 secondary antibodies (REF: A32733, LOT: RJ243415, highly cross-adsorbed, 2 mg mL^-1) referred to as “Alexa647 IgG”, Zeba Spin desalting micro columns and microscope slides were obtained from Thermo Scientific (www.thermofisher.com). Sephadex columns and Cy5-NHS activated ester were acquired from GE Healthcare (www.gelifesciences.com). Reagent solutions in DMF were prepared under argon atmosphere. All reactions took place at room temperature, if not stated otherwise. Ultrapure reagent water (resistivity > 18 MΩ cm) for buffer and solutions preparation was taken from a Milli-Q Reference system of Merck Millipore (www.merckmillipore.com).

Various reagents used in this study were purchased from the following suppliers: IQ supermix, and Agarose from Bio-Rad; DNA purification Kit using Nucleospin Extract II columns from Machery Nagel, Germany (Cat no. 740609-50); T4 Kinase Polynucleotide from Invitrogen; γ-³²P- ATP from Amersham (3000 Ci/mmol); SSDNA from Sigma Aldrich cat# D7656; Sephadex columns from GE Healthcare (Microspin G-25 column illustra 27-5325-01); and T4-Kinase from Invitrogen, Life Technology.
DNA concentration was quantified using the Gene Quant Spectrophotometer. PCR was performed using My Cycler Thermal Cycler from Bio-Rad; Membrane hybridization and crosslinking were carried out using ProBlot 12 Hybridization Oven Labnet, (31 Mayfield Avenue Edison, NJ, 08837 USA), and Spectrolinker UV Crosslinkers from Krackeler Scientific, (Inc. PO Box 1849 Albany, NY 12201-1849), respectively. Sequencing of purified DNA was carried out at the University of Saint Joseph, Department of Molecular Biology and Genetics using the Avant Genetic Analyzer (ABI 3130) machine. The sequencing reaction and subsequent purification steps were as described before [9] (link).

Protocol for antiviral efficacy was adapted from Haldar et al.^{30 (link)} and conducted at Accuratus Lab Services, MN.
Coupons were inoculated with 20 µL murine norovirus using the same procedure used for bacteria and incubated in a controlled chamber set to RH of 50% and temperature of 20 °C. After 120 min, 1.0 mL aliquots of 2× Minimum Essential Medium (Gibco Life Technologies) were pipetted individually onto each test and control coupon. The surface of each coupon was scraped with sterile plastic cell scrapers (Bioscience) and the test medium collected and passed through Sephadex columns (GE Healthcare). The filtrates were then serially diluted ten-fold and assayed for infectivity in RAW 264.7 cells.