Amicon ultra 10k device

A clear supernatant was passed through Ni–NTA column which was pre-equilibrated with a buffer (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, 5 % (v/v glycerol, 5 mM β-mercaptoethanol, 10 mM imidazole). After binding of protein, column was washed with 50 ml of washing buffer (50 mM Tris–HCl, pH 8.0, 500 mM NaCl, 5 % (v/v) glycerol, 5 mM β-mercaptoethanol, 20 mM imidazole) at 4 °C. Bound protein was eluted with 300 mM imidazole. The fractions were concentrated using Amicon Ultra 10 K device (Merck Darmstadt, Germany) and dialyzed against 50 mM Tris–HCl pH 8.0 buffer. The dialyzed sample was further loaded on Hi Trap DEAE FF (1 ml, 7 mm × 25 mm) column (GE healthcare) pre-equilibrated with 50 mM Tris–HCl buffer, pH 8.0. Bound proteins were eluted with increasing concentration of NaCl (0–1 M NaCl) in the 50 mM Tris–HCl pH 8.0 buffer. Elution was controlled by Akta purifier (GE healthcare) with a flow rate of 0.5 ml/min. CAVA eluted at 0.50 M NaCl was pooled, concentrated and stored for further study. The purity of protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

All the obtained fractions (SP, PHp and HJCp) were separated by membrane filtration in fractions containing peptides with different molecular weight ranges. All the steps were performed at 4 °C. Each sample was filtered using Amicon^® Ultra 30K device (Merck) by centrifugation at 4000× g to almost total filtration, the retentate contained compounds with MW higher than 30 kDa. The filtrates (containing compounds less than 30 kDa) were further fractionated using Amicon^® Ultra 10K device (Merck) by centrifugation at 4000× g to obtain the 10–30 kDa fraction in the retentate. Finally, the filtrates containing compounds lower than 10 kDa were centrifuged using Amicon^® Ultra 3K device (Merck) at 4000× g to obtain in the retentate fractions with 10 < MW < 3 kDa and MW < 3 kDa. Each sample was analyzed for protein content, antioxidant activity and cell culture test.

Caco-2 cells were treated with or without 2 mM IIAEK for 24 h. Following treatment, the cells were washed twice with ice-cold phosphate-buffered saline and collected in 1 mL of cold TNE buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EGTA, pH 7.5) + 1% (w/v) Triton X-100. The cell lysates were homogenized on ice using a glass homogenizer (WHEATON, 903475) with an attached potter-type shuttle (WHEATON, 358034). The homogenate was transferred to the 13PA tube (HITACHI: 332001A), and 2 mL of 80% (w/v) sucrose in TNE buffer was added. Homogenates were layered with 6 mL of sucrose density gradient solution (5–30%) using a fractionator, followed by ultracentrifugation at 200,000× g and 4 °C for 17 h in an ultracentrifuge (Himac, CP80WX, HITACHI, Tokyo, Japan) with a swing rotor (Himac, P40ST, HITACHI), as previously described [17 (link)]. Subsequently, samples were fractionated into 10 fractions and recovered using the density gradient fractionator MODEL DGF-U (HITACHI). Sucrose density gradient formation was confirmed by measuring the sucrose density (w/w %) of each fraction with the ATAGO pocket sugar meter (ATAGO). Each fraction was subjected to ultrafiltration using an Amicon^® Ultra 10K device (Millipore) according to the manufacturer’s protocol. The obtained concentrated protein solutions obtained were used for photoaffinity labeling.

The soluble recombinant His-Nme5-likeCc protein, visible after SDS-PAGE, was obtained under the following growth conditions: Escherichia coli strain BL21(DE3) harboring the plasmid was grown to OD₆₀₀ 1.2 and protein expression was induced with 0.05 mM IPTG for 1 h at 30 °C. Bacteria were collected by centrifugation, resuspended in lysis buffer (50 mM Tris HCl, 300 mM NaCl, 10 mM imidazole, and 1 mg/mL lysozyme), incubated 30 min on ice and sonicated 8 × 30 sec (50% of full power). After 25 min of centrifugation (12,000 rpm) at 4 °C, the supernatant was applied onto cobalt-charged agarose column (TALON). Histidine tagged proteins was eluted with 300 mM imidazole. Collected eluates were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA) and electrotransfered onto PVDF Hybond-P membrane (Amersham Biosciences, Buckinghamshire, United Kingdom). For the detection of His-Nme5-likeCc, the membranes were incubated with anti-His antibody (Amersham Biosciences, Buckinghamshire, United Kingdom). Protein bands were visualized using chemiluminescence detection (Amersham ECL Plus, GE Healthcare Chicago, IL, USA). Nme5-likeCc protein was concentrated and desalted using Amicon Ultra 10K device (Millipore, Burlington, MA, USA). The same Ultracel was used for buffer exchange (Nme buffer, [53 (link)]).

Conditioned media preparation assay was performed as described previously [31 (link)]. Briefly, cells were grown on 100 mm plates in about 70~ 80% confluence and then transformed to serum free media for 12 h. Cells were collected and counted, the cell ratio of different group were recorded. Conditioned media were collected after centrifugation at 2000 rpm at the temperature of 4 °C for 10 min. After being centrifuged at 12,000 rpm for 20 min, the supernatants were stored at 4 °C. For secretory proteins Western Blot assay, the supernatants were ultrafiltered with an Amicon Ultra-10 K device (Millipore) at 5000 rpm for 20 min. Concentrated media were normalized of the cell number of different groups and analyzed by Western blot analysis.