Full-length Eco RNAP (UniProt entries P0A7Z4, P0A8V2, P0A8T7, P0A800 for α, β, β’, and ω) was expressed and purified largely following the procedure as described previously 55 (link). Specifically, a pET-based plasmid harboring full length RNAP subunits α, β, ω as well as β’-PPX-His10 (PPX; PreScission protease site, LEVLFQGP, Cytiva) under an IPTG-inducible promoter (addgene #128940) was co-transformed with pACYC-Duet-1 encoding ω (addgene #128837) into Eco BL21(DE3). Shaking cultures of a total of 6 L LB media supplemented with 100 μg/ml ampicillin and 34 μg/ml chloramphenicol were inoculated with an overnight preculture from freshly transformed cells and grown to an OD600 of 1 at 37°C. Expression was induced with IPTG (0.5 mM final concentration) and cultures were incubated for 3 hr at 30°C. Cells were harvested by centrifugation (Beckman JS-4.2, 4,500×g, 4°C, 20 min) and pellets were resuspended in a total of 120 mL lysis buffer (50 mM Tris-HCl, pH 8/RT, 10 mM DTT, 1 mM ZnCl2, 5% (v/v) glycerol, 0.5x c0mplete EDTA-free protease inhibitors (Roche), 1 mM PMSF). Resuspended cells were stored at −80°C until use. Cell suspensions were thawed, lysed by high-pressure shearing (Avestin EmulsiFlex C50), and insoluble material was removed by centrifugation (Beckman JA-20, 27,000×g, 4°C, 30 min). While stirring, polyethyleneimine [PEI; 10% (w/v) in water adjusted to pH 8/RT with HCl] was slowly added to a final concentration of 0.6% (w/v) to the soluble fraction and the mixture was incubated for 25 min at 4°C while stirring. After collection of the PEI precipitate by centrifugation (Beckman JA-20, 27,000×g, 4°C, 1 hr), the pellets were washed three times in a total of 210 mL PEI wash buffer [50 mM Tris-HCl, pH 7.9/RT, 0.5 M NaCl, 5% (v/v) glycerol, 10 mM DTT]. For each wash step, the pellets were resuspended using a glass Dounce homogenizer (Wheaton) and the PEI precipitate was collected again by centrifugation. To elute RNAP from the PEI, the pellets from the last wash step were resuspended as above in a total of 120 mL PEI elution buffer [50 mM Tris-HCl, pH7.9/RT, 1 M NaCl, 5% (v/v) glycerol, 10 mM DTT] and the PEI precipitate was collected again by centrifugation saving the supernatant. Elution was repeated for a total of three rounds pooling the supernatant from each centrifugation step (final total of 360 mL). 35 g/100 mL ammonium sulfate (grounded to powder) were added slowly while stirring and the mixture was incubated overnight at 4°C. Ammonium sulfate precipitate was recovered by centrifugation (Beckman JA-18, 33000×g, 4°C, 30 min) and resuspended in a total of 50 mL IMAC buffer A [20 mM Tris-HCl, pH 7.8/RT, 1 M NaCl, 5% (v/v) glycerol, 1 mM β-mercaptoethanol]. The sample was passed through 5 μm filter and applied to 2×5 mL HiTrap IMAC HP columns (Cytiva) connected in series, charged with Ni2+ and equilibrated in IMAC buffer A. After washing the columns with increasing imidazole concentrations in buffer A (last wash with 80 mM imidazole), protein was eluted in buffer B [20 mM Tris-HCl, pH 7.8/RT, 1 M NaCl, 250 mM imidazole, 5% (v/v) glycerol, 1 mM β-mercaptoethanol]. Fractions were pooled according to protein content and His-tagged 3C protease was added at 1:36 molar ratio. The sample was dialyzed overnight at 4°C in a 12–14 kDa cutoff membrane (SpectraPor) against 20 mM Tris-HCl, pH8/4°C, 1 M NaCl, 5% (v/v) glycerol, 0.1 mM EDTA, 1 mM β-mercaptoethanol, 0.5 mM DTT. After dialysis, the sample was passed over the IMAC columns followed by dialysis of the flow-through overnight at 4°C in a 12–14 kDa cutoff membrane (SpectraPor) against 10 mM Tris-HCl, pH7.8/RT, 100 mM NaCl, 0.1 mM EDTA, 5% (v/v) glycerol, 5 mM DTT. After dialysis, the sample was loaded on a 40 mL Biorex column (Biorad Biorex-70 resin #142–5842) equilibrated in Biorex A buffer [10 mM Tris-HCl, pH7.8/RT, 0.1 mM EDTA, 5% (v/v) glycerol, 5 mM DTT] and eluted with a gradient of 20–80% Biorex B buffer [10 mM Tris-HCl, pH 7.8/RT, 1 M NaCl, 0.1 mM EDTA, 5% (v/v) glycerol, 5 mM DTT] over 15 column volumes. Fractions were pooled according to protein content. The pool was concentrated using a centrifugal filter (3,500×g, 4°C, Amicon Ultra 100K, EMD Millipore) and loaded on a Superdex 200 26/600 320 mL column (Cytiva) equilibrated in 20 mM HEPES-Na, pH 7.8/RT, 0.5 M NaCl, 0.1 mM EDTA, 5% (v/v) glycerol, 0.5 mM TCEP. Peak fractions were pooled, concentrated using centrifugal filters (3,500×g, 4°C, Amicon Ultra 100K, EMD Millipore) and mixed with buffer containing 50% (v/v) glycerol to achieve a final concentration of 20% (v/v) glycerol. Aliquots were frozen in liquid N2 and stored at −80°C until use.
Ja 20
Manufactured by Beckman Coulter
46 citations
Sourced in United States, Canada
About the product
The JA-20 is a high-speed refrigerated centrifuge designed for a wide range of laboratory applications. It features a maximum speed of 20,000 rpm and a maximum relative centrifugal force (RCF) of 48,400 x g. The JA-20 is capable of handling sample volumes up to 50 mL.
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46 protocols using «ja 20»
1
Purification of E. coli RNAP Complex
2
Purification of Eco σ^N Transcription Factor
Tagless, full-length Eco σN (RpoN, Sig54; UniProt entry P24255) was purified as described previously 21 (link). Variants of σN were obtained by cloning full-length σN into pET28a with an N-terminal His10-SUMO tag and introducing the desired mutations by PCR amplification with oligonucleotide primers carrying the desired modifications and ligation of the PCR product. Constructs were freshly transformed into Eco BL21(DE3) cells and transformants were grown at 37°C in 200 mL LB shaking cultures (baffled flasks) containing 50 ug/ml kanamycin. At OD600 of 0.6, the cultures were equilibrated to 16°C for 30 min before induction with 1mM IPTG. Expression was carried out overnight at 16°C. Cells were harvested by centrifugation (Beckman JS-4.2, 4,500 ×g, 4°C, 30 min) and resuspended in lysis buffer [20 mM Tris-HCl, pH8/RT, 500 mM NaCl, 5 mM imidazole, 5% (v/v) glycerol, 0.5 mM DTT, 1 mM PMSF, 1x c0mplete EDTA-free protease inhibitor (Roche)]. At this point, the cell resuspensions were frozen in liquid N2 and stored at −80°C until further processing. After thawing, cells were lysed by sonication (Branson Digital Sonifier 450) and subjected to centrifugation (Beckman JA-20, 11,000 ×g, 4°C, 1 hr) to remove insoluble material. The soluble fraction was passed through 0.45 μm filter and applied to a 1 mL Ni2+-charged IMAC HiTrap (Cytiva) equilibrated in buffer A [20 mM Tris-HCl, pH8/RT, 500 mM NaCl, 5% (v/v) glycerol, 0.5 mM DTT]. The column was washed with increasing concentrations of imidazole in buffer A (40 mM, 80 mM, 120 mM, 350 mM imidazole) while collecting fractions. Fractions containing Eco σN protein were pooled; His-tagged Ulp1 protease was added at 1:40 molar ratio and the sample was dialyzed overnight at 4°C against buffer A in a 12–14 kDa cutoff membrane (SpectraPor). After recovering the sample from dialysis, it was passed again over the IMAC resin to remove uncleaved fusion protein and the Ulp1 protease. The flow-through was collected, concentrated using centrifugal filters (3,500×g, 4°C, Amicon Ultra 30K, EMD Millipore) and mixed with an equal volume of storage buffer [20 mM Tris-HCl, pH8/RT, 500 mM NaCl, 40% (v/v) glycerol, 0.1 mM EDTA, 2 mM DTT]. Aliquots were frozen in liquid N2 and stored at −80°C until use.
3
Megabody Expression and Purification
The Mb9657 was constructed by grafting Nb9657 onto scaffold protein cHopQ with His6 tag on Cterminus following the protocol described elsewhere 47 . The megabody was expressed overnight in Escherichia coli WK6 strain after induction with 1 mM IPTG at OD between 2 and 3 at 600 nm. Cells were collected by a 15 min centrifugation at 5,000 rpm in Beckman JA-10 rotor. Periplasmic extract was obtained by homogenizing an equivalent of 1 l cell culture pellet in 25 mL of lysis buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 0.3 mg/ml lysozyme, 20% sucrose, 1 µg/ml leupeptin and 0.1 mg/ml AEBSF) followed by 30 min incubation at 4˚C. Next, NaCl concentration was increased to 500 mM NaCl, 5 mM MgCl2 and 50 µg/ml DNase were added, cells were centrifuged at 10,000 rpm in Beckman JA-20 rotor for 1 h and resulting supernatant was passed through 0.45 µm filter.
The periplasmic extract was then loaded on the 1 ml HisTrap HP column pre-equilibrated in 10 column volumes (CV) of wash buffer (500 mM NaCl, 100 mM Tris-HCl, 5 mM imidazole) at a flowrate of 5 ml/min, then washed by 10 CV of wash buffer and eluted in 3 CV of elution buffer (similar as wash buffer except for the imidazole concentration of 500 mM). The sample was concentrated on a spin column (10 kDa, Meck) to 1 ml and loaded on a Superdex 200 Increase 10/300 GL column equilibrated with gel filtration buffer (10 mM Tris-HCL, 140 mM NaCl, PH 7.4) and eluted with 1.2 CV. The peak fractions were collected and concentrated on a spin column (10 kDa, Meck) to 14.8 mg/ml.
The periplasmic extract was then loaded on the 1 ml HisTrap HP column pre-equilibrated in 10 column volumes (CV) of wash buffer (500 mM NaCl, 100 mM Tris-HCl, 5 mM imidazole) at a flowrate of 5 ml/min, then washed by 10 CV of wash buffer and eluted in 3 CV of elution buffer (similar as wash buffer except for the imidazole concentration of 500 mM). The sample was concentrated on a spin column (10 kDa, Meck) to 1 ml and loaded on a Superdex 200 Increase 10/300 GL column equilibrated with gel filtration buffer (10 mM Tris-HCL, 140 mM NaCl, PH 7.4) and eluted with 1.2 CV. The peak fractions were collected and concentrated on a spin column (10 kDa, Meck) to 14.8 mg/ml.
Corresponding organizations : Vrije Universiteit Brussel, Victoria University of Bangladesh
4
Nanobody Purification and Expression
The nanobody repertoire was cloned into phage-display vector pMESy4 which allows for expression of Nbs with a 6xHis-tag. Overexpression of Nbs was done overnight after induction by 1 mM Isopropyl β-d-1-thiogalactopyranoside (IPTG) of Escherichia coli WK6 strain after the cell density reached OD of 0.8 at 600 nm. Cells are collected by a 15 min centrifugation at 5,000 rpm in Beckman JA-10 rotor. Cells were lysed by adding 4 mL of lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mg/ml lysozyme, 20% sucrose, 50 µg/ml DNase, and 1 mM phenylmethylsulphonyl fluoride (PMSF)] per 1 g of pellet followed by 30 min incubation at 4˚C. Periplasmic extract was obtained by 15 min centrifugation at 10,000 rpm in Beckman JA-20 rotor. MgCl2 (5 mM) was added to the extract, the extract was filtered, supplemented with 20% sucrose, and stored at -80˚C in aliquots.
Corresponding organizations : Vrije Universiteit Brussel, Victoria University of Bangladesh
5
Recombinant Nanobody Expression
The Nb repertoire was cloned into phage-display vector pMESy4, which allows for the expression of Nbs with a 6xHis tag and EPEA tag. Overexpression of Nbs was done overnight after induction by 1 mM IPTG of Escherichia coli WK6 strain after the cell density reached OD of 0.8 at 600 nm. Cells are collected by a 15 min centrifugation at 5000 rpm in Beckman JA-10 rotor. Cells were lysed by adding 4 ml of lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1 mg/ml lysozyme, 20% sucrose, 50 μg/ml DNase, and 1 mM phenylmethylsulphonyl fluoride] per 1 g of pellet followed by 30 min incubation at 4 °C. Periplasmic extract was obtained by 15 min centrifugation at 10,000 rpm in Beckman JA-20 rotor. MgCl2 (5 mM) was added to the extract, the extract was filtered, supplemented with 20% sucrose, and stored at −80 °C in aliquots.
Top 5 most cited protocols using «ja 20»
1
Cell Fractionation and In Vitro Translation
S30 extracts were prepared as described in (16 (link)). Briefly, Krebs-2 ascites cells were collected in a centrifuge tube with isotonic buffer (150 mM NaCl, 35 mM Tris–HCl pH 7.5), and four times washed with centrifugation (Beckman JA-20, 1200 rpm, 10 min) in equal volumes of the same buffer. After the fourth centrifugation, cells were re-suspended in 1.5 volumes of hypotonic buffer (10 mM Tris–HCl pH 7.5, 10 mM KCH3COO, 1.5 mM Mg(CH3COO)2, 2.5 mM DTT) and incubated on ice for 20 min. Then the cells were disrupted using a Dounce homogenizer (type B pestle, 15–20 strokes), and the lysate was centrifuged for 20 min at 15 000 rpm. The supernatant was aliquoted and stored at –75°C. Translation experiments were performed in a total volume of 10 μl, which contained 5 μl of the S30 extract, translation buffer (20 mM Hepes–KOH pH 7.6, 1 mM DTT, 0.5 mM spermidine–HCl, 0.6 mM Mg(CH3COO)2, 8 mM creatine phosphate, 1 mM ATP, 0.2 mM GTP, 120 mM KCH3COO and 25 μM of each amino acid), 2 units of Human Placental Ribonuclease Inhibitor (HPRI, Fermentas) and 0.25 pmol mRNA (or mRNA mix), for 1 h. The luciferase activities were measured using the Dual Luciferase Assay kit. Translation in nuclease treated RRL (Promega) was performed exactly as suggested by the manufacturer, with the addition of 25 μM of l -methionine (instead of radiolabel [35S]methionine). When indicated, various amounts of m7GTP (equilibrated with respective amount of Mg(CH3COO)2) were added, and the translation mix was pre-incubated for 5 min before the addition of mRNA. For experiments with 4E-BP1, the recombinant protein was used. Plasmid pGEX-6p1-h4E-BP1 (a gift from Y. Svitkin and N. Sonenberg, McGill University, Montreal) encoding human 4E-BP1-GST fusion was expressed in Escherichia coli, and 4E-BP1 was purified using Glutathione-Sepharose 4B and PreScission Protease (Amersham) and dialyzed against buffer A100 (20 mM Tris–HCl pH 7.5, 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol). Highly purified 4E-BP1 protein was added to the translation mix in a volume less than 1.5 μl and incubated for 5 min before the addition of reporter mRNAs. In the control, an equal amount of A100 buffer was added.
Corresponding organizations : Lomonosov Moscow State University, Engelhardt Institute of Molecular Biology
2
Protein Purification of His-Tagged Mutants
BL21(DE3) cells transformed with a pET28 vector carrying each mutant gene were grown in 2 L of LB medium containing 50 mg/L kanamycin at 37 °C until the OD600 reached ∼0.6. The incubation temperature was then changed to 18 °C, and the culture was induced with 0.2 mM IPTG. The induced cells were shaken continually at 18 °C for an additional 18 h. The cells were harvested by centrifugation (11900g for 10 min, Beckman JLA-10.500 rotor). The cell pellet was resuspended in 30 mL of start buffer [50 mM HEPES·Na (pH 7.4), 300 mM NaCl, and 10% glycerol, containing a protease inhibitor cocktail from Roche Applied Science] and stored at −80 °C.
All purification steps were performed at 4 °C. Cell paste in start buffer was sonicated on ice for 20 min. After centrifugation (23700g for 30 min, Beckman JA-20 rotor), the supernatant was filtered through a 0.45 μm syringe filter. The sample was then loaded onto a 5 mL HisTrap HP column (GE Healthcare Life Sciences). The column was washed with 20 mL each of start buffer containing 25 and 50 mM imidazole and then 10 mL each of start buffer containing 100, 200, and 500 mM imidazole. The eluent was collected in several fractions, which were analyzed by Tris-SDS−PAGE. The fractions containing the desired protein (200−500 mM imidazole) were combined and concentrated using an Amicon Ultra-15 centrifugal filter unit (30 kDa molecular mass cutoff for His6-VenL, 10 kDa molecular mass cutoff for other proteins, Millipore) to less than 2 mL. Buffer exchange of the concentrated protein with start buffer was conducted twice using a PD-10 desalting column (GE Healthcare Life Sciences). The resulting protein sample was aliquoted and stored at −80 °C. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology).
All purification steps were performed at 4 °C. Cell paste in start buffer was sonicated on ice for 20 min. After centrifugation (23700g for 30 min, Beckman JA-20 rotor), the supernatant was filtered through a 0.45 μm syringe filter. The sample was then loaded onto a 5 mL HisTrap HP column (GE Healthcare Life Sciences). The column was washed with 20 mL each of start buffer containing 25 and 50 mM imidazole and then 10 mL each of start buffer containing 100, 200, and 500 mM imidazole. The eluent was collected in several fractions, which were analyzed by Tris-SDS−PAGE. The fractions containing the desired protein (200−500 mM imidazole) were combined and concentrated using an Amicon Ultra-15 centrifugal filter unit (30 kDa molecular mass cutoff for His6-VenL, 10 kDa molecular mass cutoff for other proteins, Millipore) to less than 2 mL. Buffer exchange of the concentrated protein with start buffer was conducted twice using a PD-10 desalting column (GE Healthcare Life Sciences). The resulting protein sample was aliquoted and stored at −80 °C. Protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology).
Corresponding organizations : University of Illinois Urbana-Champaign, Center for Genomic Science, Illinois College
3
Paclitaxel-Loaded PLGA Nanoparticles
PLGA NS were prepared by solvent displacement followed by polymer deposition [18 (link)]. PTX (3% w/w) and 75 mg PLGA NS were dissolved in 20 ml acetone. The organic phase was poured dropwise (0.5 ml/min), under magnetic stirring, into 40 ml of a water/ethanol solution (1:1, v/v) containing 0.5% (w/v) Tween 80 to obtain a milky colloidal suspension. The organic solvent was then evaporated off under high vacuum at 40°C. The different formulations were purified from untrapped PTX and unadsorbed surfactant by means of centrifugation (15000 × g) for 1 h at 5°C, using a Beckman (Fullerton, CA) J2-21 model centrifuge equipped with a Beckman JA-20.01 fixed-angle rotor. The supernatants were discarded and the pellets were resuspended in 50 ml of water, then centrifuged using the same conditions described above. The entire operation was repeated 3 times. After final washing, the NS were resuspended in 5 ml of filtered water (0.22-μm Sartorius membrane filters) in the presence of 5% of HP-Cyd as a cryoprotector [18 (link)] and freeze-dried. Freeze-dried NS were resuspended with filtered water and were characterized for size distribution, surface chemistry and technological parameters. For the in vitro studies, freeze-dried NS were resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine and 100 U/ml penicillin-streptomycin.
Coumarin 6-loaded NS were prepared similarly for the fluorescent microscopic studies. The polymer solution contained 0.05% (w/v) coumarin-6 as fluorescent marker instead of PTX.
Coumarin 6-loaded NS were prepared similarly for the fluorescent microscopic studies. The polymer solution contained 0.05% (w/v) coumarin-6 as fluorescent marker instead of PTX.
Corresponding organizations : University of Catania
4
Purification of CUL3-RBX1 and RhoBTB3-CUL3-RBX1 Complexes
GST–Cyclin E was expressed and purified as the binary complex but without detergent. For purification of a CUL3–RBX1 binary complex, GST-CUL3 and 2HA-RBX constructs were co-transformed into BL21 (DE3) Rosetta II cells and cultures (OD600 = 0.6) were induced with 0.1 mM IPTG for 18 h at 16°C. Cells were resuspended in 50 mM Hepes, pH 7.4, 300 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.5% Triton X-100, and full protease inhibitor cocktail and lysed by two passes at >10,000 lb/in2 in an EmulsiFlex-C5 apparatus (Avestin). Lysates were clarified by centrifugation twice at 20,000 rpm for 20 min each in a rotor (JA 20; Beckman Coulter). Clarified lysates were incubated with glutathione 4B–Sepharose beads (GE Healthcare) for 1 h at 4°C, washed with 50 vol of 50 mM Hepes, pH 7.4, 300 mM NaCl, 1 mM DTT, and 5 mM MgCl2, and eluted with 50 mM Hepes, pH 7.4, 300 mM NaCl, 1 mM DTT, 5 mM MgCl2, and 20 mM glutathione. For in vitro reconstitution of RhoBTB3–CUL3–RBX1 complexes, a FLAG-RhoBTB3 construct was transformed into BL21 (DE3) Rosetta II cells, and cultures (OD600 = 0.6) were induced with 0.1 mM IPTG for 18 h at 16°C. Cells were resuspended in 50 mM Hepes, pH 7.4, 300 mM NaCl, 1 mM DTT, 5 mM MgCl2, 0.5% Triton X-100, and full protease inhibitor cocktail and lysed by two passes at >10,000 lb/in2 in an EmulsiFlex-C5 apparatus (Avestin). Lysates were clarified by centrifugation twice at 20,000 rpm for 20 min each in a rotor (JA 20; Beckman Coulter). Clarified lysates were incubated with anti-FLAG M2 antibody–conjugated agarose (Sigma-Aldrich) for 2 h at 4°C, and then washed with 50 vol of 50 mM Hepes, pH 7.4, 300 mM NaCl, 1 mM DTT, and 5 mM MgCl2. After this, excess CUL3–RBX1 binary complex was mixed with purified FLAG-RhoBTB3 bound to anti-FLAG antibody–conjugated agarose for 90 min at 4°C followed by a 50-vol wash with 50 mM Hepes, pH 7.4, 300 mM NaCl, 1 mM DTT, and 5 mM MgCl2 to remove unbound CUL3–RBX1. Finally, RhoBTB3–CUL3–RBX1 ternary complexes were eluted with 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM DTT, 1 mM MgCl2, and 0.2 mg/ml FLAG peptide (Sigma-Aldrich). Stoichiometry of purified binary and ternary complexes was analyzed by SDS-PAGE, Coomassie blue staining, and immunoblotting. Proteins were stored on ice.
Corresponding organizations : Stanford University
5
Purification and Characterization of BDCA-2 CRD
Expression of the wild type CRD from human BDCA-2 following published protocols for other C-type CRDs resulted in inclusion bodies that were isolated as described (21 (link)). Inclusion bodies from 2 liters of bacterial culture were dissolved in 30 ml of 6 m guanidine HCl containing 100 mm Tris-Cl (pH 7.8) and incubated in the presence of 0.01% (v/v) 2-mercaptoethanol for 30 min at 4 °C. Following centrifugation for 30 min at 100,000 × g in a Beckman Ti70.1 rotor, the supernatant was diluted dropwise into 120 ml of 0.5 m NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2 at 4 °C, followed by dialysis against 2 changes of 2 liters of the same buffer. Insoluble material was removed by centrifugation for 30 min at 50,000 × g in a Beckman JA20 rotor, and the supernatant was applied to a 5-ml column of glycopeptide-agarose. After rinsing with 12 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2, the bound protein was eluted with 10 × 1-ml aliquots of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 2.5 mm EDTA. Fractions containing the CRD were identified by analyzing aliquots on SDS-polyacrylamide gels, with protein detected by staining with Coomassie Blue.
Mutant forms of the CRD were expressed in the same way, but following initial dialysis against the renaturation buffer, the proteins from 4–6 liters of culture were further dialyzed against two changes of 2 liters of H2O and lyophilized. The lyophilized proteins were taken up in 6 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2 and centrifuged at 100,000 × g in a Beckman TLA100.4 rotor for 30 min at 4 °C. The supernatant was applied to a 10-ml column of mannose-Sepharose, which was washed five times with 2-ml aliquots of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 25 mm CaCl2 and eluted with three 2-ml aliquots and eight 1-ml aliquots of 50 mm NaCl, 25 mm Tris-Cl, pH 7.8, and 2.5 mm EDTA.
Gel filtration was performed on a 1 × 30-cm Superdex 200 column (GE Healthcare) eluted with 10 mm Tris-Cl (pH 7.8), 100 mm NaCl, and 2.5 mm EDTA at a flow rate of 0.5 ml/min, with absorbance monitored at 280 nm. Gel electrophoresis was performed on SDS-polyacrylamide gels containing 17.5% (w/v) acrylamide.
Mutant forms of the CRD were expressed in the same way, but following initial dialysis against the renaturation buffer, the proteins from 4–6 liters of culture were further dialyzed against two changes of 2 liters of H2O and lyophilized. The lyophilized proteins were taken up in 6 ml of 150 m
Gel filtration was performed on a 1 × 30-cm Superdex 200 column (GE Healthcare) eluted with 10 m
Corresponding organizations : Imperial College London, Stanford University
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