Seakem gold

Conditioned media was treated with proteinase K, and HA was then precipitated with equal volume of 100% ethanol and resuspended in deionized H₂O. Processed samples were separated on 0.4% agarose gel (SeaKem Gold, Lonza, Allendale, NJ, USA) in 1x tris acetate EDTA buffer (100V for 75 minutes) with ice blocks to maintain gel temperature, followed by a Southern blot transfer process to HyBond+ membrane in 100 mM tris acetate, pH 7.3 for 16 hours. HA was visualized on the membrane by western blot analysis. Specifically, 2% milk in phosphate-buffered solution (PBS) 0.05% Tween 20 was used as blocking reagent and as reagent diluent. Instead of using a primary antibody, biotin-TSG6-ΔHep-Fc (39) (0.5 µg/ml) was used to bind HA molecules on the membrane and horseradish peroxidase (HRP)-conjugated streptavidin was used for detection. To demonstrate that the sample processing procedure did not affect HA size, Select-HA 1000K (Hyalose, Oklahoma City, OK, USA) was spiked into conditioned media prior to sample processing. The specificity of biotin-TSG6-ΔHep-Fc was demonstrated by an HA blot of conditioned media that was pre-treated with hyaluronidase, Streptomyces hyaluronlyticus nov. species (1 unit/300 µl culture media, 37°C for 1 hour; EMD Millipore, Burlington, MA, USA).

SmaI (New England Biolabs Inc., MA, USA) restriction enzyme patterns of the clinical isolates and reference strains were investigated by PFGE according to a published protocol for Streptococcus pneumoniae [11 ], with minor modifications. Bacterial suspensions of 8.0–9.0 McFarland density were embedded in SeaKem Gold (Lonza, Rockland, ME, USA) agarose. The electrophoresis run time was 16 h. SYBR Safe (Invitrogen, CA, USA) was used to stain the DNA fragments and GelCompar II software (version 6.6 Applied Maths NV, Belgium) was used to examine the PFGE patterns. Analysis was conducted using the unweighted pair grouping method with arithmetic mean (UPGMA) using the Dice similarity coefficient, and optimization and position tolerance were both set at 1.5%. Clonal clusters were determined by using an 85% similarity cut-off [12 (link)]. ATCC 49619 Streptococcus pneumoniae was used to root a phylogenetic tree. Based on the PFGE analysis, representative isolates from each cluster, including their sub-clusters, were selected for further sequence analysis of 16S rRNA and rpoB genes.

Large plasmids were extracted as described previously [34 (link)]. The aqueous phase containing the plasmids was analyzed using 0.6% agarose gel (Seakem Gold, Lonza, Quakertown, PA, USA, Catalog: 50152) with 1 × TBE buffer in a precooled electrophoresis chamber at a voltage of 10 v/cm for 4–5 h. The gel was stained with 1 μg/mL of ethidium bromide for 15 min and destained in demineralized water at 4 °C overnight before imaging.