This was performed using DEAE-Sephacel (Sigma-Aldrich, Burlington, Massachusetts, USA) column equilibrated in 0.05 M Tris-HCl, 0.01 M NaCl, pH 7.4 (Tris buffer) at 10˚C. The column was eluted using a linear gradient of 0.01–0.8 M NaCl in Tris buffer. The absorbance of eluted proteins was monitored at 230 nm (Holochrome monitor). Fractions (LKB fraction collector) were collected (2.4 ml), and those containing DMP1 were identified using Dot blotting.
Deae sephacel
Manufactured by Merck Group
Sourced in United States
DEAE-Sephacel is a versatile ion-exchange chromatography media used for the purification and separation of biomolecules. It consists of diethylaminoethyl (DEAE) functional groups covalently attached to a cross-linked agarose matrix, providing a high-capacity anion exchanger. The core function of DEAE-Sephacel is to facilitate the selective adsorption and elution of charged biomolecules, enabling their efficient purification from complex mixtures.
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Lab products found in correlation
Potassium hydroxide, by Thermo Fisher Scientific (1 mentions)
Ammonium sulfate, by Thermo Fisher Scientific (1 mentions)
Tricine, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Pepstatin, by Merck Group (1 mentions)
Thiamine pyrophosphate, by Merck Group (1 mentions)
Acetic acid, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Hepes free acid, by Merck Group (1 mentions)
Bamhi restriction enzyme, by New England Biolabs (1 mentions)
Succinyl coa, by Merck Group (1 mentions)
Glycine, by Thermo Fisher Scientific (1 mentions)
Centricon concentrator, by Merck Group (1 mentions)
Sds page reagents, by Thermo Fisher Scientific (1 mentions)
Pyridoxal 5 phosphate, by Merck Group (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
Ampicillin, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
9 protocols using deae sephacel
1
DEAE-Sephacel Protein Purification
2
Purification and Characterization of Enzymes
3
Chitinase Concentration and Purification
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For concentration of the chitinase centrifugal device of 3 kD size (Centrifugal Devices, PALL corporation, USA) were used. Culture supernatant was added to the upper chamber of the centrifugal device and centrifuged at 1500 g for 3–4 h. 750 ml of culture supernatant was concentrated 10 times.
The concentrated enzyme extract was then subjected to ion exchange chromatography with DEAE Sephacel and DEAE Sepharose (Sigma-Aldrich, USA) separately. After loading the enzyme concentrate, 2 ml of flow through was collected in 10 fractions in micro centrifuge tubes. Column was washed again with 10 ml of 50 mM Tris HCl, pH 7 without NaCl. Five fractions of 2 ml aliquots were collected and designated as wash. The enzyme bound in column was eluted with a step wise increase of NaCl concentration in 5 ml with 100, 200, 300, 400, 500, 600 and 700 mM NaCl. Total 30 fractions of 2 ml each were collected and stored at 4 °C until further use. The eluted fractions were then checked for total protein estimation (Bradford method) and chitinase enzyme activity at 585 nm. The fractions were also checked on SDS PAGE (by Coomasie Brilliant Blue).
The concentrated enzyme extract was then subjected to ion exchange chromatography with DEAE Sephacel and DEAE Sepharose (Sigma-Aldrich, USA) separately. After loading the enzyme concentrate, 2 ml of flow through was collected in 10 fractions in micro centrifuge tubes. Column was washed again with 10 ml of 50 mM Tris HCl, pH 7 without NaCl. Five fractions of 2 ml aliquots were collected and designated as wash. The enzyme bound in column was eluted with a step wise increase of NaCl concentration in 5 ml with 100, 200, 300, 400, 500, 600 and 700 mM NaCl. Total 30 fractions of 2 ml each were collected and stored at 4 °C until further use. The eluted fractions were then checked for total protein estimation (Bradford method) and chitinase enzyme activity at 585 nm. The fractions were also checked on SDS PAGE (by Coomasie Brilliant Blue).
4
Protein Purification and Characterization
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Carbon-coated copper grid was purchased from Ted Pella Inc (Redding, CA). DNase, DEAE-Sephacel, (−)-Epigallocatechin gallate (EGCG), isopropylthiogalactoside (IPTG), lysozyme, 2-(N-morpholino)ethanesulfonic acid (Mes), 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-oxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), Sephacryl S-200, S-Sepharose, and thioflavin-T (Th-T) were purchased from Sigma-Aldrich (St. Louis, MO). 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) was obtained from Avanti Polar Lipids Inc (Alabaster, AL). LB medium was purchased from Becton Dickinson & Company (San Diego, CA), and uranyl acetate was obtained from Electron Microscopy Sciences (Hatfield, PA). Microcon YM30 was provided by Merck Millipore (Billerica, MA). Dimethyl cyclodextrin was supplied from the Microbial Carbohydrate Resource Bank at Konkuk University.
5
Purification of RIG-I and T7 RNA Polymerase
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Human RIG-I was expressed using pET28 SUMO vector in Escherichia coli strain Rosetta (DE3) (Novagen). The cell lysate soluble fraction was purified using a Ni2+-nitrilotriacetate (QIAGEN) column, followed by Ulp1 SUMO protease digestion to remove 6xHis-SUMO tag. It was further purified by hydroxyapatite (CHT-II, Bio-Rad) and heparin Sepharose column chromatography (GE Healthcare). Purified protein was dialyzed into 50 mM HEPES pH 7.5, 50 mM NaCl, 5mM MgCl2, 5 mM DTT, and 10% glycerol overnight at 4°C, then snap frozen in liquid nitrogen and stored at −80°C (Jiang et al., 2011 (link)).
T7 RNA polymerase was expressed using pAR1219 vector in Escherichia coli strain BL21 (Novagen). Cell lysate was purified in three steps: SP-Sephadex (Sigma Aldrich), CM-Sephadex (Sigma Aldrich), and then DEAE-Sephacel (Sigma Aldrich). Finally, purified protein was dialyzed into Storage Buffer (20 mM sodium phosphate pH 7.7, 1 mM Na3-EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 50% (v/v) glycerol) (Jia and Patel, 1997 (link)).
T7 RNA polymerase was expressed using pAR1219 vector in Escherichia coli strain BL21 (Novagen). Cell lysate was purified in three steps: SP-Sephadex (Sigma Aldrich), CM-Sephadex (Sigma Aldrich), and then DEAE-Sephacel (Sigma Aldrich). Finally, purified protein was dialyzed into Storage Buffer (20 mM sodium phosphate pH 7.7, 1 mM Na3-EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 50% (v/v) glycerol) (Jia and Patel, 1997 (link)).
6
Determining HA Molecular Weight in Brain
7
Glucose-Dependent TGF-β Signaling Assay
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Dulbecco’s Modified Eagle medium (DMEM) (0 mM and 25 mM glucose) was from GIBCO BRL, Grand Island, USA. SB431542 and DEAE-sephacel were purchased from Sigma-Aldrich, MO, USA. GKA (RO28-1675) was from Axon Medchem, The Netherlands. Anti-rabbit IgG HRP, GAPDH, anti-phospho-Smad2 (Ser465/467) rabbit monoclonal antibody and human transforming growth factor beta-1 (TGF-β) was from Cell Signalling Technology, Danvers, USA. Glucokinase activator Compound A was from Merck, Darmstadt, Germany. [35S]-sulfate was from MP Biomedicals, Irvine, CA. Cetylpyidinium chloride (CPC) was from Uni-Lab Chemicals and Pharmaceuticals, India. 3MM Whatman chromatography paper was from Whatman International, Ltd., Maldstone, UK. YSI 2300 Stat plus glucose and lactate analyser kindly provided by Professor Stephen Bird, Exercise and Metabolism Research Laboratory, RMIT University. YSI buffer concentrated kit, YSI glucose standard were from YSI Inc, Yellow Spring, USA.
8
Purification of Asen via IEC and Spray Drying
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IEC separation was performed at room temperature on DEAE Sephacel (Sigma Aldrich, St. Louis, Mo) column (54 × 20 cm). The column was firstly equilibrated with degassed water. Dissolved Asen was loaded and eluted by water (around 10 L) at a flow rate of 40 mL min -1 to obtain a first fraction called IEC-F1. Subsequently, a gradient was performed during 5 h starting with water and finishing with 2 M NaCl (flow rate: 20 mL min -1 ). A second fraction termed IEC-F2 was then obtained, eluting with 2 M NaCl (around 20 L), at a flow rate of 20 mL min -1 . The volume employed during the gradient phase was added to the 20 L corresponding to the fraction IEC-F2. The two fractions obtained were separately heated (50 °C) and concentrated using a cross flow filtration system (ÄKTA flux, GE Healthcare) (flow: 4 L min -1 ; TMP -corresponding to feed pressure -Retentate pressure-: 15 psi; column: UFP-30-C-4x2MA, 30 kg mol -1 ). Fractions IEC-F1 and IEC-F2 were afterward diafiltered against 10 volume of water in the same conditions to eliminate salts. The fractions were spray-dried using a B-290 Mini Spray Dryer (BUCHI™).
9
Isolation and Reconstitution of Photosynthetic Reaction Centers
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Carotenoid-less Rb. sphaeroides R-26 cells were grown photoheterotrophically under anaerobic conditions in medium supplemented with potassium succinate (Ormerod et al. 1961) (link). Chromatophores and RCs were prepared as described earlier (Tandori et al. 1995) (link). RCs were solubilized by DDAO (N,N-dimethyldodecylamine N-oxide; Fluka) and purified by ammonium sulfate precipitation followed by DEAE-Sephacel (Sigma) anion-exchange chromatography.
In order to obtain RCs with full and known Q B site quinone activity for herbicide titration experiments, proteins were prepared by deprivation of native ubiquinone-10 (UQ-10) and subsequent reconstitution through addition of the desired amount of UQ-10. The quinone depletion was carried out according to the method of Okamura (Okamura et al. 1975) (link).
Deprived RCs were concentrated by centrifuge filter (Whatman VectaSpin 3, 10 kDa exclusion) to 80-100 µM. The fractions with OD280/OD803 ratio between 1.27 and 1.50 were collected and used for further experiments. For optical spectroscopy, the RC suspension was diluted to 1 μM in detergent suspension (10 mM TRIS, 100 mM NaCl, 0.01% DDAO, pH 8.0 buffer). The Q B site of RCs was reconstituted with native UQ-10 addition just before herbicide titration experiments (see below).
In order to obtain RCs with full and known Q B site quinone activity for herbicide titration experiments, proteins were prepared by deprivation of native ubiquinone-10 (UQ-10) and subsequent reconstitution through addition of the desired amount of UQ-10. The quinone depletion was carried out according to the method of Okamura (Okamura et al. 1975) (link).
Deprived RCs were concentrated by centrifuge filter (Whatman VectaSpin 3, 10 kDa exclusion) to 80-100 µM. The fractions with OD280/OD803 ratio between 1.27 and 1.50 were collected and used for further experiments. For optical spectroscopy, the RC suspension was diluted to 1 μM in detergent suspension (10 mM TRIS, 100 mM NaCl, 0.01% DDAO, pH 8.0 buffer). The Q B site of RCs was reconstituted with native UQ-10 addition just before herbicide titration experiments (see below).
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