Calcium standard for icp

Polyethersulfone (PES) microfiltration membranes (0.45 μm pore diameter, 47 mm diameter) were purchased from MilliporeSigma (Burlington, USA). Glycidyl methacrylate (≥97.0%), lanthanum(III) nitrate hexahydrate (99.999% trace metal basis), cerium(III) chloride heptahydrate (99.9% trace metal basis), neodymium(III) nitrate hexahydrate (99.9% trace metal basis), zinc perchlorate hexahydrate, calcium chloride (anhydrous, Redi-Dri, ≥97%), sodium standard for ICP (1,000 mg/L ± 2 mg/L), calcium standard for ICP (1,000 mg/L ± 2 mg/L), and magnesium standard for ICP (1,000 mg/L ± 2 mg/L) were purchased from Sigma-Aldrich. Magnesium chloride hexahydrate (99–102%, ACS grade) was purchased from VWR Life Science. L-lysine (98%, powder) was purchased from Alfa Aesar. Ethyl alcohol (absolute, 200 proof, ≥99.5%, ACS Reagent grade) was purchased from Acros Organics. Denatured Ethyl alcohol (88–91%), sodium chloride (≥99%, certified ACS grade, crystalline), nitric acid (68–70%, certified ACS plus grade), and hydrochloric acid (36.5–38%, certified ACS plus grade) were purchased from Fisher Chemical. Arsenazo (III) was purchased from Pointe Scientific. Sodium hydroxide (10 N, biotech reagent grade) was purchased from Avantor. Deionized water (DI water) was made from a RiOS-DI 3 water purification system (MilliporeSigma, Burlington, MA, USA).

The calcium content of the SiiE_Cterm variants was analysed by inductively coupled plasma—atom emission spectroscopy. All proteins were purified as described above, but without the second Glutathione Sepharose step after tag cleavage. The purified SiiE_Cterm proteins were concentrated and dialysed against the same batch of buffer (5 mM Tris-HCl, pH 8.0, ratio of sample-to-buffer volume 1:100). Calcium standard for ICP (Sigma-Aldrich) was diluted to 0.5, 5 and 50 μg x ml^-1 with the same buffer. The ICP-AES analyses were performed using a Ciros CCD (Spectro Analytical Instruments GmbH). Three individual measurements of the same sample were conducted for each variant and the mean calculated.