Mouse uncarboxylated osteocalcin was purified from BL21 transformed with pGEX2TK-mOCN as described (Lee et al., 2007 (link); Mera et al., 2016 (link)). Briefly, GST-osteocalcin fusion protein was produced in BL21 pLyS transformed with pGEX2TK-mOCN after induction with IPTG. Cells were collected in lysis buffer (PBS 1X, 10mM Tris pH 7.2, 2mM EDTA, 1% Triton and 1X protease and phosphatase inhibitor cocktail (Thermo, 78443). Following 4 freeze/thaw cycles and sonication, lysates were cleared by centrifugation. The supernatant was incubated with glutathione-sepharose 4B (GE, 17075601) for 4 hours at 4 °C. Following 6 washes with washing buffer (PBS 1X, 1% Triton) and with PBS 1X, osteocalcin was then cleaved out from the GST moiety by using thrombin (GE, 27-0846-01). Four fractions were collected and each of them was incubated with Benzamidine sepharose (GE, 17-5123-10) for 30 minutes at room temperature to remove thrombin.
Benzamidine sepharose
Manufactured by GE Healthcare
Benzamidine Sepharose is a laboratory equipment used for affinity chromatography. It consists of benzamidine, a chemical compound, covalently coupled to Sepharose, a type of agarose-based beads. This product is designed to isolate and purify serine proteases, a class of enzymes, from various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin, by GE Healthcare (3 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Vent dna polymerase, by New England Biolabs (2 mentions)
Nadph, by AppliChem (2 mentions)
E coli er2566 competent cells, by New England Biolabs (2 mentions)
Gsh sepharose4 fast flow, by GE Healthcare (2 mentions)
Sephacryls 200 resin, by Cytiva (2 mentions)
N methyl mercaptoacetamide, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Quickchange site directed mutagenesis kit, by Agilent Technologies (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Hydrogen peroxide, by Thermo Fisher Scientific (1 mentions)
Cary 50 uv vis spectrophotometer, by Agilent Technologies (1 mentions)
Thrombin, by Merck Group (1 mentions)
Glutathione sepharose 4b resin, by GE Healthcare (1 mentions)
Chitin agarose beads, by New England Biolabs (1 mentions)
Hydrogen peroxide 30 solution, by Thermo Fisher Scientific (1 mentions)
L selenocystine, by Thermo Fisher Scientific (1 mentions)
5 protocols using benzamidine sepharose
1
Purification of Mouse Uncarboxylated Osteocalcin
2
Purification of Uncarboxylated Osteocalcin
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Mouse uncarboxylated OCN was purified from BL21 transformed with pGEX2TK-mOCN as previously described (Lee et al., 2007 (link); Ferron et al., 2010a (link); Oury et al., 2011 (link), 2013 (link); Mera et al., 2016 (link)). In brief, GST-OCN fusion protein was bacterially produced in BL21 pLyS transformed with pGEX2TK-mOCN after induction with IPTG. Cells were collected in lysis buffer (PBS 1×, 10 mM Tris, pH 7.2, 2 mM EDTA, 1% Triton, and 1× protease and phosphatase inhibitor cocktail; 78443; Thermo Fisher Scientific). Following four freeze-thaw cycles and sonication, lysates were cleared by centrifugation. The supernatant was incubated with glutathione-Sepharose 4B (17075601; GE) for 4 h at 4°C. Following six washes with washing buffer (PBS 1× and 1% Triton) and with PBS 1×, OCN was then cleaved out from the GST moiety by using thrombin (27-0846-01; GE). Four fractions were collected, and each of them was incubated with benzamidine Sepharose (17-5123-10; GE) for 30 min at room temperature to remove thrombin.
3
Constructing PfTR Mutants via Mutagenic PCR
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All mutagenic polymerase chain reaction (PCR)
primers utilized in the construction of PfTR mutants were purchased
from Integrated DNA Technologies (Coralville, IA). The Quickchange
site-directed mutagenesis kit was purchased from Agilent Technologies
(Santa Clara, CA). Vent DNA polymerase, NdeI, SapI, T4 DNA ligase, E. coli ER2566 competent cells, and chitin-agarose beads
were purchased from New England Biolabs (Ipswich, MA). GSH-sepharose
4 Fast Flow, benzamidine sepharose, and 2′-4′-ADP-sepharose
resins were obtained from GE Healthcare (Waukesha, WI). Sephacryl
S-200 resin was purchased from Amersham Pharmacia (Uppsala, Sweden).
NADPH was obtained from AppliChem (Darmstadt, Germany). N-(Methyl)mercaptoacetamide, βME, anddl -α-lipoic
were from Sigma-Aldrich (St. Louis, MO). Bovine thrombin was purchased
from BioPharm Laboratories (Bluffdale, UT).l -Selenocystine
was from Acros Organics (Morris Plains, NJ). Hydrogen peroxide (30%
solution) and EDTA were purchased from Fisher Scientific (Fair Lawn,
NJ). All enzyme kinetic assays were performed on a Cary50 UV–vis
spectrophotometer (Cary, Walnut Creek, CA) and were conducted at
room temperature.
primers utilized in the construction of PfTR mutants were purchased
from Integrated DNA Technologies (Coralville, IA). The Quickchange
site-directed mutagenesis kit was purchased from Agilent Technologies
(Santa Clara, CA). Vent DNA polymerase, NdeI, SapI, T4 DNA ligase, E. coli ER2566 competent cells, and chitin-agarose beads
were purchased from New England Biolabs (Ipswich, MA). GSH-sepharose
4 Fast Flow, benzamidine sepharose, and 2′-4′-ADP-sepharose
resins were obtained from GE Healthcare (Waukesha, WI). Sephacryl
S-200 resin was purchased from Amersham Pharmacia (Uppsala, Sweden).
NADPH was obtained from AppliChem (Darmstadt, Germany). N-(Methyl)mercaptoacetamide, βME, and
were from Sigma-Aldrich (St. Louis, MO). Bovine thrombin was purchased
from BioPharm Laboratories (Bluffdale, UT).
was from Acros Organics (Morris Plains, NJ). Hydrogen peroxide (30%
solution) and EDTA were purchased from Fisher Scientific (Fair Lawn,
NJ). All enzyme kinetic assays were performed on a Cary50 UV–vis
spectrophotometer (Cary, Walnut Creek, CA) and were conducted at
room temperature.
4
Purification of GST-tagged Proteins
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Pelleted cells were resuspended and lysed in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 [pH 7.4]). 500 μL Glutathione Sepharose 4B resin (GE Healthcare) was used for clarified cell lysate from 1 L bacterial culture. The resin was incubated with clarified lysate for 2 h at 4 °C, followed by washing with 50 bed vol of PBS, then 50 bed vol of PBS with 100 mM NaCl, and lastly with 50 bed vol of PBS. To remove the GST tag, resin was incubated with 2 bed vol of PBS containing 10 μL of 1 U/μL thrombin (Sigma) for 2 h at 25 °C with occasional agitation. To remove thrombin, 100 μL Benzamidine-Sepharose (GE Healthcare) was then added, and the eluate was incubated for 30 min at 4 °C with rocking. Glycerol was added to the eluates at 10% (v/v) final concentration for storage at −80 °C.
5
Construction and Characterization of PfTR Mutants
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