Hitrap deae ff

AKTAprime plus (GE Healthcare) was used for the fractionation of erythrocyte proteins. In the case of gel filtration chromatography, the erythrocyte lysate was fractionated by using HiPrep 16/60 Sephacryl Columns S-300 HR (GE Healthcare) equilibrated with 20 mM Tris–HCl (pH 7.4) containing 150 mM NaCl. For ion exchange chromatography, oxDJ-1 immunoreactive fractions were applied to a HiTrap DEAE FF (GE Healthcare) equilibrated with 20 mM Tris–HCl (pH 7.4) containing 150 mM NaCl. Proteins were eluted with a NaCl gradient (150–700 mM).

Lactobacillus gasseri (BCRC14619) was cultured in Lactobacillus MRS broth (Merck Millipore, Burlington, MA, USA) at 37 °C, according to the ATCC guidelines. The pelleted bacteria were first dissolved in 1% lysozyme (Sigma-Aldrich, St. Louis, MO, USA) solution to lyse the cells. Ammonium sulfate (Sigma-Aldrich) was then added incrementally to the cytosolic fraction. The precipitated crude extracts were then separated through DEAE-Sepharose ion-exchange chromatography (HiTrap™ DEAE FF; GE Healthcare, Chicago, IL, USA) and further sub-separated through Sephacryl S-300 HR size-exclusion chromatography (Amersham Biosciences, Buckinghamshire, UK). The crude extracts, fractions, and sub-fractions were subsequently co-cultured with mouse bone marrow-derived dendritic cells (BMDC) to screen the active ingredient (detailed information is provided in Additional file 1).

Tinospora cordifolia plant was obtained from Ch. Devi Lal Rudraksh Vatika Herbal Nature Park, Bhudkalan, Yamunanagar, Haryana, India. The bark of green stem was removed by scalpel blade and homogenized in 50 mM Tris–HCl pH 8.0 with 500 mM NaCl. All reagents of highest purity grade were purchased from Sigma–Aldrich (St. Louis, MO, USA), Merck (Darmstadt, Germany) and GE Healthcare. Hi Trap DEAE FF and Superdex-200 columns were purchased from GE Healthcare, Uppsala, Sweden. Electrophoresis reagents were purchased from the Bio-Rad Laboratories (Richmond, CA, USA). Databases used are http://www.matrixscience.com/, http://www.uniprot.org., http://blast.ncbi.nlm.nih.gov/Blast.cgi and http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d2//.

Recombinant plasmids pET32a-S100A4 were transferred into E. coli BL21 (DE3) and induced to express the S100A4 protein by isopropyl-β-d-thiogalactopyranoside (IPTG). Following this, the cell lysate was sonicated and centrifuged. Protein samples from the supernatant, precipitation and cell lysate were analyzed by SDS-PAGE and stained with Coomassie brilliant blue to confirm the pET32a-S100A4 expression. The supernatant sample was purified by ion exchange chromatography column (HiTrap™ DEAE FF; loading buffer: Tris-HCl 0.02mol/l, pH 8.8; and elution buffer: 50mM/100mM/200mM/500mM NaCl Tris-HCl 0.02mol/l, pH 8.8; GE Healthcare Life Sciences, Chalfont, UK) according to the instructions of the manufacturer. Protein concentration was measured with a Bradford protein assay kit (Boster, Wuhan, China) using bovine serum albumin (BSA; Huasheng Biotech, Inc., Tianjin, China) as a standard (10 (link)).

Secreted syndecan EDs were purified from HUVEC as in previously described protocols^{69 (link),70 (link)}. Briefly, 10–12 tissue culture dishes (10 cm diameter) of confluent HUVEC were transduced with adenovirus expressing the indicated syndecan ED (MOI = 5–10) in serum-free, growth factor-added media (EGM-2 MV). Media plus one PBS wash were collected at 36 and 72 h post-transduction. Each fraction was kept frozen until purification. Syndecan-rich media (~300 ml) was filtered on 0.45 µm filter unit (NALGENE) and then pass through a 1 ml DEAE column (GE Healthcare HiTrap^™ DEAE FF) using a peristaltic pump (flow ~1.5 ml/min). The column was then washed with 10 column volumes of PBS, 10 column volumes PBS (0.25 NaCl), followed by elution with 20 column volumes of phosphate-buffered 2 M NaCl. Samples were buffer exchanged to 150 mM NaCl, concentrated to ~6 ml and adjusted to pH ~7.2. To this solution 250 µL of anti-HA conjugate agarose resin (ThermoFisher #26181) was added and incubated for 16–18 h at 4 °C under gentle rotation. The resin was then washed 4 times with PBS and batch-eluted 4 times with 500 µl of a 3 N NaSCN solution. Buffer was exchanged to low PBS (20 mM NaCl, 10 mM phosphate buffer, pH = 7.4) and concentrated to ~150 µl. Purified EDs were used for HS compositional analysis.

The established purification procedure for recombinantly produced human m-calpain comprises several chromatographic steps and was performed as described by Hata et al. (2012) with some minor modifications as follows: (1) 0.3 mM AEBSF was used as a protease inhibitor in the lysis buffer instead of 0.3 mM PMSF; (2) the DEAE-toyopearl and the MonoQ HR10/10 anion exchange columns were substituted by a Hitrap DEAE FF and a MonoQ 4.6/100 PE anion exchange columns (GE Healthcare), respectively.

Total RNA was extracted from rose petals using the RNeasy Plant Mini Kit (Qiagen). cDNA of RyPPDC was amplified by RT-PCR using the 3′-RACE method with degenerate primers [TaKaRa RNA PCR Kit (AMV) Ver.3.0, TaKaRa]. 5′-RACE was performed with specific primers using the Smarter Race cDNA Amplification Kit (TaKaRa). The RyPPDC proteins expressed in insect cells were collected and washed in PBS45 (link). The proteins were extracted with 100 mM Tris–HCl buffer (pH 8.0) containing 1% TritonX-100, 1 mM TPP, and 5 mM MgCl₂. The recombinant proteins were subjected to further purification by HiTrap DEAE FF, HiTrap Phenyl HP, and Superdex 200 10/300 GL (GE Healthcare).