Amicon ultra 4 10k

Before immunoaffinity isolation of oxidized ApoA-I, IgG (Fc) heavy chains were first depleted from human plasma as follows. IgG (Fc) depletion column (GWB-IGGIGY, Genway Biotech) was washed with 1 mL of 0.01% NaN₃/PBS solution 5 times. A 500 μL aliquot of plasma samples was applied to the column and then applied for a second time followed by an additional wash with 0.01% NaN₃/1 mL PBS. The collected through-flow fraction (1.5 mL) was then subsequently applied on anti-LDL–Sepharose 4B (supplied by MONA Ltd.) and then on an HDL depletion column (GWB-HDLIGY, Genway Biotech). The depletion columns were washed with PBS as above. The collected flow-through fraction was run 3 times through the HDL depletion column for maximum protein binding and purity. Bound proteins were stripped off from the columns by washing with 2 mL of 1× stripping buffer (0.1 M glycine/HCl, pH 2.5). After addition of 200 μL of 1 M Tris, pH 8.0, directly to the collected flow-through fraction, samples were concentrated with Amicon Ultra-4 10K (MilliporeSigma) by centrifugation at 1,700g for 30–40 minutes at 4°C to a final volume of 150 μL.

The culture supernatant of the Candida biofilm was centrifugally fractionated into solutions of <10 kDa and >10 kDa. Briefly, Candida supernatants were put into an ultrafiltration column (Amicon Ultra-4 10K, Merck KGaA) and centrifuged at 6000× g for 20 min. The flow-through solution was used as <10 kDa fraction, and the concentrate fraction inside the column was used as >10 kDa fraction. The >10 kDa fraction was diluted with RPMI 1640 supplemented with MOPS medium before use. E. coli biofilms were formed in the presence of each supernatant fraction and 50 μg/mL MEPM at 37 °C for 24 h. The control wells without supernatant fractions contained an equivalent amount of DMSO. The E. coli CFUs were counted as described above.

The purified (His)₈-tagged MOX-1 was concentrated to 10 mg/mL with Amicon Ultra-4 10 K (Merck Millipore Ltd.), and it was subjected to crystallization. Crystals for X-ray analysis were grown at 16°C by the hanging-drop vapor diffusion method with reservoir solutions of 14–20% polyethylene glycol 8,000 in 100 mM sodium cacodylate buffer (pH 6.5) with 0.2 M zinc acetate. Crystals were soaked with S02030 by dipping in a reservoir solution containing 5–10 mM S02030 for 10–60 min. These crystals were flash frozen in a liquid nitrogen stream (100°K) with 20% ethylene glycol as a cryoprotectant and immediately subjected to data collection at Station 5A of the Photon Factory, the High Energy Accelerator Research Organization (KEK, Ibaraki, Japan). The reflection was indexed and integrated using iMosflm and SCALA in CCP4 package (Collaborative Computational Project Number 4, 1994 (link)). The initial model for molecular replacement was the structure of free MOX-1 (PDB entry: 3W8K). Molecular replacement and refinement were performed with MOLREP in CCP4 (Vagin and Teplyakov, 2010 (link)). The coordinate and structure factor files have been deposited in the Protein Data Bank under the accession code 5ZYB.

Trypsin activated Cry1Ia protein was biotinylated by using the protein biotinylation kit from GE Healthcare (GE Healthcare, Little Chalfont, UK), as described elsewhere [66 (link)]. The eluted fractions were quantified by NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA), analyzed by 12% SDS-PAGE and verified by Western blot. The protein fractions were concentrated by using an Amicon Ultra-4 10K centrifugal filter device (Merck Millipore, Tullagreen, Ireland) and stored at 4 °C.
The interference of biotin in oligomerization was tested following incubation of the biotin labelled Cry1Ab with O. nubilalis BBMV. The detection of biotin labelled Cry1Ab oligomerization was performed following the same protocol than has been described for Cry1Ia. The results showed no influence of biotin in oligomerization (Figure S1), as had been already claimed by other authors that have used biotin labelled proteins to detect oligomers [12 (link),20 (link),53 (link),54 (link)].