Enzytec oxalic acid

2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), bulk CuCO₃·Cu(OH)₂, CuSO₄, TBA and TTM were purchased from Sigma Aldrich, while agar, malt extract and potassium chloride from VWR (Oxoid, Darmstadt, Germany).
The oxalic acid assay Enzytec oxalic acid was purchased from R-Biopharm AG. The silver stain kit, Dodeca Silver Stain was obtained from BIO-RAD.
Two commercial aqueous suspensions of MCA were investigated. The two MCA formulations contain comparable amounts of Cu particles but differ in the amount of TBA: MCA_HTBA contained 5% w/w and MCA_LTBA 0.4% w/w TBA.

After incubation, 0.5 mL of liquid media were taken from each culture medium and oxalate concentration in the samples was analyzed with a spectrophotometer (Genesys 10S UV-vis, Thermo Scientific Inc., Waltham, MA, USA) at 590 nm as specified by the instruction manual (Enzytec Oxalic acid, R-Biopharm AG). Samples were diluted 100-fold with distilled water before being used in the assay as they showed very high activities.
To determine any further changes in the protein secretions of R. placenta 45 exposed to the different amended media after incubation, 0.2 mL of liquid media was taken from each culture medium. Protein extracts from the control, CuCO₃·Cu(OH)₂, CuSO₄, MCA liquid media -with and without TTM- plus markers (BIO-RAD) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels loaded with 5 μL of each liquid culture sample and the marker. Gels were silver stained with the Dodeca Silver Stain kit according to the instruction manual (BIO-RAD).