For the purification of EGFP-conjugated Fv fragments, VH-SARAH-EGFP and VL-SARAH were co-transfected into HEK293T cells at a 1 : 1 molar ratio using a Lipofectamine 3000 Transfection Kit (ThermoFisher Scientific). EGFP conjugated nanobodies (Nb-EGFPs) were transfected into HEK293 cells using the same kit. Culture supernatants were collected 4–5 days after the transfection. Antibody fragments in the supernatants were collected with Ni-NTA agarose (QIAGEN) at room temperature for 2 hours, washed three times with ice-cold Tris Buffered Saline (TBS; 20 mM Tris, 150 mM NaCl, pH 7.5) and once with TBS containing 10 mM imidazole (Nacalai Tesque, Inc). Bound antibody fragments were eluted with 200 mM imidazole in TBS. The purified polypeptides were dialyzed vs. TBS using Spectra/por membrane (MWCO: 6–8 kD, Spectrum Laboratories, Inc.), with two exchanges of buffer overnight at 4°C. Transiently transfected HEK293T cells in a 10 cm dish typically yield 0.34–4.02 μg (5–60 pmol) Fv-EGFP and 0.41–4.92 μg (10–120 pmol) Nb-EGFP, which are enough for 20–60 times of IRIS imaging. The purified probes were mostly fluorescent because the concentration measured by fluorescent intensity was comparable to the concentration determined by CBB protein assay.
Imidazole
Manufactured by Nacalai Tesque
Sourced in Japan
Imidazole is a heterocyclic organic compound with the chemical formula C3H4N2. It is a colorless, crystalline solid that serves as a core functional group in various chemical and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (3 mentions)
Ni nta agarose, by Qiagen (1 mentions)
Gateway system, by Thermo Fisher Scientific (1 mentions)
Pd 10 gel filtration column, by GE Healthcare (1 mentions)
V 530 spectrophotometer, by Jasco (1 mentions)
Ampicillin, by Nacalai Tesque (1 mentions)
2 14c malonyl coa, by PerkinElmer (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Glycine, by Nacalai Tesque (1 mentions)
Sodium sulfate, by Nacalai Tesque (1 mentions)
L arginine, by Nacalai Tesque (1 mentions)
L ascorbic acid, by Nacalai Tesque (1 mentions)
Fucoidan from fucus vesiculosus, by Merck Group (1 mentions)
5 protocols using imidazole
1
Purification of EGFP-conjugated Fv Fragments
2
Expression and Purification of His-tagged zFGF2
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His-tagged zebrafish-fgf2 (zFGF2) protein was expressed in Sf9 insect cells using a Gateway system (Invitrogen), according to the manufacturer’s instructions, followed by purification using imidazole (19004-22, Nacalai, Japan)-affinity chromatography.
3
Characterization of PHCP Protein Redox States
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The PHCP wild‐type protein (final 6 μM) in 10 mM potassium phosphate buffer (pH 7.0) was air‐oxidized or reduced with a grain of sodium dithionite. The reduced PHCP protein solutions were bubbled with N2 (95.0%), and then with CO (99.9%, GL Science, Japan) in the dark. The same reduced PHCP solutions bubbled with N2 were loaded on a PD‐10 gel filtration column (GE Healthcare) equilibrated with 10 mM potassium phosphate buffer (pH 7.0) in order to remove excess sodium dithionite, and were then mixed with NONOate (diethylamine sodium salt hydrate, Sigma‐Aldrich; final 5 mM) in 10 mM NaOH (pH 8.0), which released NO gas under the neutral pH conditions, in the dark to determine whether or not the NO gas was bound. The air‐oxidized and reduced PHCP solutions were also mixed with 20 mM imidazole (Nacalai Tesque, Japan) and 0.5% (v/v) butyl‐isocyanide (Tokyo Chemical Industry, Japan), respectively. Visible absorption spectra of these preparations were obtained with a JASCO V‐530 spectrophotometer (JASCO, Japan) at 25°C.
4
Enzymatic Synthesis of Polyphenols
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The THC and p-coumaroyl-CoA were purchased from TransMIT (Gießen, Germany). Malonyl-CoA, naringenin, trans-p-coumaric acid, resveratrol, ampicillin, imidazole, and isopropyl 1-β-D-thiogalactoside were purchased from Nacalai Tesque (Kyoto, Japan). Acetonitrile and trifluoroacetic acid were obtained from Sigma-Aldrich Japan (Tokyo, Japan). Bis(sulfosuccinimidyl) suberate disodium salt was obtained from Dojindo Laboratories (Kumamoto, Japan), whereas Ni2+-coated magnetic beads (His Mag Sepharose Ni) and Ni2+-coated Sepharose beads (His SpinTrap) were purchased from GE Healthcare Japan. All chemicals were of analytical grade except for Acetonitrile and trifluoroacetic acid, which were of HPLC grade. [2-14C]Malonyl-CoA (1480 MBq/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA, USA).
5
Sulfated Carbohydrates and Chemicals Analysis
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D-(+)-Galactose (Gal), L-arginine, glycine, barium chloride dihydrate, L-ascorbic acid, sodium sulfate, and imidazole were obtained from Nacalai Tesque Inc. (Kyoto, Japan). Galactose-6-O-sulfate (G6S), neocarrabiose-4-O-sulfate (NB4S), neocarratetraose-41,3-di-O-sulfate (NTdS), 3β-galactobiose (β-Gal-[1→3]-Gal) (GalB), and 3α-4β-3α-galactotetraose (α-Gal-[1→3]-β-Gal-[1→4]-α-Gal-[1→3]-Gal) (GalT) were purchased from Dextra Laboratories Ltd. (Shinfield, UK); all sulfated chemicals were in the form of sodium salts. D-(+)-Galacturonic acid monohydrate, piperazine hexahydrate, and 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid (TSP-d4) were purchased from Wako Pure Chemical Co. (Osaka, Japan). Deuterium oxide (D2O, 99.8 atom% D), 1-(2-pyrimidinyl) piperazine, and sodium rhodizonate were obtained from Kanto Chemical Co. Inc. (Tokyo, Japan).
Fucoidan from Fucus vesiculosus was purchased from Sigma-Aldrich Co. (≥95% pure, Cat. No. F8190, St. Louis, MO, USA).
Fucoidan from Fucus vesiculosus was purchased from Sigma-Aldrich Co. (≥95% pure, Cat. No. F8190, St. Louis, MO, USA).
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