I use double distilled water to make an azocasein solution (2%) but it does not dissolve easily and needs to vortex for over 15 minutes. Moreover, this milky solution precipitates after one day. Is this precipitation usual? Or is this because of degeneration of azocasein? I conducted an assay with this substrate and obtained unexpected results (not considerable difference between blank and treatment).
Azocasein can be dissolved in distilled water (up to ~5 mg/cm3, i.e. ~0.5%). For higher concentrated aqueous solutions it is convenient to select a suitable alkaline buffer solution, since it is known to precipitate from aqueous solution at acid pH. For instance, to prepare 100 cm3 of a 2 wt% azocasein solution in sodium carbonate ─ hydrogen carbonate buffer, we could proceed as follows: (i) prepare Na2CO3 solution at 0.1 M ─ dissolve 10.599 g of Na2CO3 in H2O and dilute to 1 dm3; (ii) prepare NaHCO3 solution at 0.1 M ─ dissolve 8.401 g of NaHCO3 in H2O and dilute to 1 dm3; (iii) prepare 50 cm3 of Na2CO3 ─ NaHCO3 buffer solution of pH ≈ 9.2 (20 ºC) by adding 5 cm3 of Na2CO3 0.1 M to 45 cm3 of NaHCO3 0.1 M ─ the pH can be modified by adding different volumes if required; (iv) dissolve 2.000 g of azocasein in previous solution at 60 ºC while stirring but avoiding air entrainment; (v) let the solution cool to room temperature while stirring and further dilute to complete 100 cm3 by adding H2O; (vi) keep the solution refrigerated if not required for immediate use.
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