pET-28-vector encoded, His6-tagged Plk1 WT, S529A, T539A, 3 A, 3D, or Sgo1 were expressed individually in E. coli BL21 DE3 (Novagen). We lysed bacteria in lysis buffer (50 mM Na2HPO4, 100 mM NaCl, 10% glycerol, 10 mM imidazole), added Ni2 + -NTA-agarose (Qiagen), and washed beads with washing buffer (50 mM Na2HPO4, 100 mM NaCl, 10% glycerol, 20 mM imidazole). Corresponding proteins were purified from Ni2 + -NTA-agarose (Qiagen) according to standard procedures.
Ni2 nta agarose
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, China
Ni2+-NTA agarose is a solid-phase affinity chromatography resin used for the purification of proteins with histidine-tags. It consists of nickel-nitrilotriacetic acid (Ni2+-NTA) immobilized on agarose beads. The Ni2+ ions bind to histidine residues on the target protein, allowing it to be captured and separated from other components in the sample.
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Lab products found in correlation
Glutathione sepharose 4b, by GE Healthcare (4 mentions)
Protein a g agarose bead, by Beyotime (3 mentions)
Hiload 26 60 superdex 200 pg, by GE Healthcare (3 mentions)
Hiload 16 60 superdex 200 pg column, by GE Healthcare (2 mentions)
Dntps, by Takara Bio (2 mentions)
Anti cleaved caspase 9, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti senp6 hpa024376, by Merck Group (2 mentions)
Anti cleaved parp, by Cell Signaling Technology (2 mentions)
Glutathione sepharose 4b, by Cytiva (2 mentions)
Pqe30, by Qiagen (2 mentions)
Gsh sepharose, by Cytiva (2 mentions)
Optima xpn 80, by Beckman Coulter (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Xl 90 ultracentrifuge, by Beckman Coulter (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Duchefa Biochemie (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
97 protocols using ni2 nta agarose
1
Purification of Recombinant Plk1 and Sgo1
2
Purification of BCL10 and MALT1 Proteins
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pET21aBCL10-His was transformed into BL21(DE3) E. coli cells. Protein expression was induced with 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) for 4 hr at 37°C. E. coli cells were lysed in lysis buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM imidazole, 8 M urea), sonicated. The lysates were centrifuged at 13k rpm (kUBOTA 1920) for 10 min at 4°C. The soluble fraction was applied to Ni2+ NTA agarose (Qiagen). Purification was performed as the manufacture’s instruction. The protein was eluted with 250 mM imidazole.
pET21a-MALT1-His or pET21a-MALT1_C464A-His was transformed into Arctic-ExpressTM RIL compent E. coli cells. Protein expression was induced with 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) for 48 hr at 8°C. E. coli cells were suspended in buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM imidazole) and lysed by 700 psi french press (Thermo IEC FRENCH press laboratory with mini pressure cell, 120VAC, 60Hz). The lysates were centrifuged at 13k rpm (kUBOTA 1920) for 10 min at 4°C. The soluble fraction was applied to Ni2+ NTA agarose (Qiagen). The protein was purified according to the manufacture’s instruction and eluted with 250 mMimidazole.All the purified proteins were dialyzed against PBS and stored at –70°C freezer in the presence of 20% glycerol.
pET21a-MALT1-His or pET21a-MALT1_C464A-His was transformed into Arctic-ExpressTM RIL compent E. coli cells. Protein expression was induced with 1 mM IPTG (isopropyl β-D-thiogalactopyranoside) for 48 hr at 8°C. E. coli cells were suspended in buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 10 mM imidazole) and lysed by 700 psi french press (Thermo IEC FRENCH press laboratory with mini pressure cell, 120VAC, 60Hz). The lysates were centrifuged at 13k rpm (kUBOTA 1920) for 10 min at 4°C. The soluble fraction was applied to Ni2+ NTA agarose (Qiagen). The protein was purified according to the manufacture’s instruction and eluted with 250 mMimidazole.All the purified proteins were dialyzed against PBS and stored at –70°C freezer in the presence of 20% glycerol.
3
Expression and Purification of VHH-B5 Protein
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The pET22b VHH-B5 plasmid vector was transformed into the BL21 Rosetta strain (Novagen, USA) and protein expression was induced at OD600 = 0.8 with 1 mM IPTG at 18°C for 16 hours. Bacterial pellets were stored at -80°C and lysed by incubation in 25 mM Tris pH 7.4/150 mM NaCl/5 mM imidazole/0.5% Trition-C100/20 mM MgCl2 containing 0.25 mg/mL Lysozyme, DNaseI 40 U/mL and 2 mM PMSF at room temperature. The insoluble fraction was spun down by centrifugation at 6000 × g for 45 min. The soluble fraction was then incubated with Ni2+-NTA-Agarose (QIAGEN) for 45 min at room temperature and washed with 25 mM Tris pH 7.4/150 mM NaCl/5 mM imidazole. Protein was eluted with the same buffer containing 500 mM imidazole and was then further purified by size exclusion chromatography (SEC) on a HiLoad 16/60 Superdex 200 pg column (GE Healthcare Bio-Sciences AB, Sweden) with a flow rate of 1 mL/min at 4°C.
4
Purification of His-tagged ASRT Proteins
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Overnight cultures of E. coli BL21 that contain the plasmid for the production of his-tagged ASRT proteins were induced with 0.8 mM IPTG (Duchefa, Netherland) for 4 h at 35 ℃. Induced E. coli cells were harvested and suspended in sonication buffer (50 mM TrisHCl pH 7.0, 150 mM NaCl). Cells were lysed with 0.5 mM PMSF (phenylmethylsulphonyl fluoride, USB, USA) by sonication (Branson sonifier 250) at 4 ℃ followed by low-speed (3,220 × g for 20 min) centrifugation (Eppendorf centrifuge 5810R) to remove cell debris. Cell lysates were then sedimented at 95,000 × g for 1 h at 4 ℃ (Ti70 rotor, Beckman XL-90 ultracentrifuge), and the supernatant was transferred to a new tube. The supernatant was incubated with Ni2+-NTA agarose (Qiagen) and purified with imidazole38 (link).
5
Recombinant scFv Antibody Production
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In total, 102 recombinant single-chain fragment variable (scFv) antibodies (Table S1 ), stringently selected from an in-house-designed phage display library, were produced in 100 mL Escherichia coli cultures. In brief, the antibodies were purified from the cell supernatant using affinity chromatography on Ni2+-NTA agarose (Qiagen, Hilden, Germany) and eluted in 250 mM imidazole. The buffer was changed to PBS by extensive dialysis, and the antibodies were stored at 4 °C until used for microarray production. The protein concentration was determined by measuring the absorbance at 280 nm, and the degree of purity and integrity of the scFv antibodies was verified with 10% SDS-PAGE (Invitrogen, Carlsbad, CA, USA).
6
SUMOylation Affinity Purification of ANXA1
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SUMOylated ANXA1 in 293T cells was affinity-purified under denaturing conditions by Ni2+-NTA pull down. Briefly, HA-ANXA1 and His-SUMOs (SUMO1, SUMO2, and SUMO3) were overexpressed in HEK293T cells with Flag-tagged Ubc9. After 24 hours, cells were first washed twice with cold PBS and then lysed in 800 μl of Ni2+-NTA denaturing buffer [6 M Gu-HCl, 10 mM tris, 100 mM NaH2PO4 (pH 8.0), and 20 mM N-Ethylmaleimide (NEM)]. After shearing the DNA by sonication (2 × 20 s), the samples were cleared by centrifugation (15,000g, 10 min, 4°C). The supernatants were mixed with 50 μl of prewashed Ni2+-NTA agarose (Qiagen) and incubated for 3 hours at 4°C on a rotating wheel. The beads were then washed in successive washing steps in 1 ml of Ni2+-NTA washing buffer [8 M urea, 10 mM tris-HCl (pH 6.3), 100 mM NaH2PO4, and 0.1% Triton X-100] and lastly eluted by boiling the beads in 50 μl of 2× SDS-PAGE loading buffer containing 200 mM imidazole at 95°C for 5 min. Bound proteins were resolved by SDS-PAGE and detected by immunoblot analysis.
7
Autophagy Regulation Pathway Assay
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The antibodies used in the present study are listed in table S1. Protein A+G agarose beads, LPS, recombinant murine IFN-γ, IL-4, and LysoTracker Red were obtained from Beyotime Biotechnology (Haimen, China), and the Ni2+-NTA agarose was purchased from QIAGEN (Dusseldorf, Germany). CHX was obtained from Calbiochem (508739; Darmstadt, Germany). Dimethyl sulfoxide (DMSO; D2650), MG-132 (C-2211), ammonium chloride (NH4Cl; A9434), 3-MA (M9281), CQ phosphate (PHR1258), and puromycin (P9620) were purchased from Sigma-Aldrich, China. Rapamycin (HY-10219) was obtained from MedChemExpress (Shanghai, China), Bafilomycin A1 (S1413) was purchased from Selleck (Houston, TX, USA), and EBSS was purchased from Gibco (Gaithersburg, MD, USA). A set of small interfering RNA of control (6568) and BECN1 (6246) was purchased from Cell Signaling Technology (Danvers, MA, USA). All other general reagents are commercially available and used as received.
8
Purification and Biochemical Characterization of Sirtuins
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All restriction enzymes, DNA-modifying enzymes, and DNA ladders were obtained from New England Biolabs. Plasmid pETM-41 and TEV protease were kindly provided by Dr Amit Sharma (ICGEB, New Delhi). Protein markers were obtained from Thermo Fisher Scientific (USA). nicotinamide Adenine Dinucleotide [Adenylate-32P] (800Ci/mmol) was purchased from American Radiolabeled Chemicals (USA). Ni2+-NTA agarose and amylose resin were purchased from Qiagen and New England Biolabs, respectively. Sirtinol, nicotinamide, cambinol, and Ex-527 were obtained from Sigma-Aldrich (USA). SIRT1 Fluorometric Drug Discovery Kit and SIRT5 Fluorometric Drug Discovery Kit were procured from Enzo Life Sciences (USA). Other materials used in this study were of analytical grade and were commercially available.
9
Purification of His-tagged Nitrogenase Proteins
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His-tagged NITs in pET-21b(+) (Novagen) were expressed at 37 °C in E. coli BL21-CodonPlus (DE3)-RIL (Agilent Technologies) for 4 h in LB containing 100 μg ml−1 ampicillin and 1 mM IPTG. The cells were pelleted at 4 °C and resuspended in 50 mM Tris–Hcl pH 8.0, 100 mM NaCl with cOmpleteTM protease inhibitor (Roche) and sonicated on ice for 4 min (at 15 s intervals) (Sonicator® 3000, Misonix). The clarified supernatant was loaded onto a Ni2+-NTA-agarose (Qiagen) column and washed with 50 mM Tris–Hcl pH 8.0, 500 mM NaCl, 20 mM imidazole and eluted over a gradient up to 500 mM imidazole. The NIT-containing peak was pooled and loaded onto a TSKgel PWXL4000 HPLC size exclusion column (Tosoh Corp), equilibrated with 50 mM Tris–Hcl pH 8.0, 100 mM NaCl and the leading edge of the NIT-containing peak was collected. Cryo-EM grids were immediately prepared using this material and aliquots destined for long-term storage were flash frozen in liquid nitrogen and stored at −80 °C after addition of 1 mM DTT. Activity assays were performed after buffer exchange into 50 mM potassium phosphate pH 8.0, 1 mM DTT.
10
Reconstitution and Purification of RNA Polymerase Holoenzyme
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