Cell pellets were resuspended in Buffer A (50 mm Tris, 200 mm NaCl, pH 7.5, in Milli-Q water) supplemented with complete EDTA-free protease inhibitor mixture (Roche Applied Science), lysozyme, DNase, and RNase (Sigma). The cells were lysed on ice using a Bandelin Sonoplus sonicator with a TT13/F2 tip, set to 30% power, 20 s on, 40 s off for 30 min. Cellular debris was removed by ultracentrifugation using a Ti50.2 rotor in a Beckman Optima LE-80k ultracentrifuge at 40,000 rpm for 1 h at 4 °C. The supernatant was passed through a 0.45-μm filter. The clarified supernatant was applied to a gravity flow nickel-nitrilotriacetic acid-agarose column (Qiagen). The column was washed with three column volumes of Buffer A supplemented with 10 mm imidazole and then three column volumes of Buffer A supplemented with 40 mm imidazole. Protein was eluted in 1-ml fractions using Buffer A supplemented with 250 mm imidazole. Fractions containing the purified protein were buffer-exchanged into Buffer B (20 mm Tris, 100 mm NaCl, pH 7.5, in Milli-Q water) using a 10DG column (Bio-Rad). Protein aliquots were flash-frozen until required.
Nickel nitrilotriacetic acid agarose column
Manufactured by Qiagen
Sourced in Spain
The Nickel-nitrilotriacetic acid agarose column is a laboratory equipment used for protein purification. It is designed to bind and purify proteins with a histidine-tag. The column is packed with agarose beads coated with nickel-nitrilotriacetic acid, which can selectively bind to the histidine-tag on the target protein, allowing it to be separated from other proteins in the sample.
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Lab products found in correlation
Sonoplus sonicator, by Bandelin (1 mentions)
10dg column, by Bio-Rad (1 mentions)
Complete edta free protease inhibitor mixture, by Roche (1 mentions)
Optima le 80k, by Beckman Coulter (1 mentions)
Dnase, by Merck Group (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Pd 10 column, by Cytiva (1 mentions)
5 protocols using nickel nitrilotriacetic acid agarose column
1
Purification of His-tagged Recombinant Proteins
2
Recombinant APE1/Ref-1 Protein Purification
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Human full length APE1/Ref-1 DNA was inserted into the pET28b expression vector (Novagen, Gibbstown, NJ), containing a 6-histidine tag for easy purification [10 (link)]. pET28b-APE1/Ref-1 plasmids were then transformed into the BL21(DE3) strain of Escherichia coli. Following induction with isopropyl β-D-1-thiogalactopyranoside (IPTG), the cells were sonicated in lysis buffer (100 mM NaCl, 20 mM HEPES), and the recombinant protein was purified on a nickel-nitrilotriacetic acid agarose column (Qiagen, Valencia, CA). After washing, the isolated APE1/Ref-1 protein was eluted with 250 mM imidazole buffer followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Liverpool, UK) in PBS, and frozen in 10% glycerol at −80°C. This protein was subsequently used for the standard curve in the APE1/Ref-1 ELISA.
3
Purification of Recombinant EiGS1-1 Protein
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The EcoRI and NotI tags were created on the 5ʹ and 3ʹ ends of the coding region of EiGS1-1-WT and EiGS1-1-R59 cDNA, which was fused with the His6 tag at the C-terminal end. The amplified PCR products were subcloned into the pPic9k vector and sequenced. The correct pPic9k-EiGS1-1-WT and pPic9k-EiGS1-1-R59 plasmids were linearized and transformed into Pichia pastoris strain GS115 competent cells, which were grown on BMGY medium plates. Positive clones were detected by PCR using the primer pair given in Table 2 . Clones with high positive expression were selected and identified by SDS-PAGE. His-tagged protein in transgenic yeast was affinity purified using a nickel-nitrilotriacetic acid agarose column (Qiagen). The protein peak fractions were analysed for the presence of a single band of recombinant EiGS1-1 protein (~40 kDa) on the polyacrylamide gel and stained with Coomassie Blue (Supplementary Fig. S1 ).
4
Recombinant Production and Purification of HTB
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HTB (GenBank: AJY26992.1) was identified in a cDNA library prepared from H. irritans salivary glands. The production of recombinant HTB was performed as described [18 (link)]. Briefly, the sequence coding for the predicted mature HTB was amplified by PCR from a recombinant plasmid carrying the full-length cDNA derived from an oligo-capped full-length enriched library prepared from the H. irritans salivary gland mRNA. The fragment was amplified with primers carrying sites for KpnI and SalI and cloned into a KpnI/SalI-cut pQE-30 vector (Qiagen, Hilden, Germany). The plasmid was used to transform Escherichia coli M15 cells, and protein expression was induced using 0.1 mM isopropyl-D-thiogalactopyranoside for 2 h at 37 °C. The N-terminal 6× histidine-tagged HTB was purified using a nickel-nitrilotriacetic acid agarose column (Qiagen) under denaturing conditions following the manufacturer’s instructions. Antigenic similarity between native and recombinant proteins was confirmed by Western blot using anti-HTB polyclonal rabbit sera blotted against H. irritans salivary gland extract (Additional file 1 : Figure S1). The alignment was performed by MUSCLE [19 (link)] and graphically edited using BioEdit software [20 ].
5
Constructing pRSET-His-IM30 Plasmid
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To construct the pRSET-His-IM30 plasmid, the im30 gene (sll0617) from Synechocystis was amplified by PCR using the two primers 5'im30 and 3'im30 (Table S1) and genomic Synechocystis DNA as a template (Fuhrmann et al., 2009b) . PCR product as well as the plasmid pRSET (Schoepfer, 1993) were restriction-digested with NdeI and BamHI and subsequently ligated (Fuhrmann et al., 2009b) . The plasmids used for expression of the IM30 variants IM30_AAL (Hennig et al., 2015) , IM30_FERM (Heidrich et al., 2016) encoding the respective amino acids were mutated to code for alanine. The naturally occurring alanine residues were mutated to serine. The oligonucleotides used for mutation are listed in Table S1.
Purification of IM30 E. coli cells were broken by ultrasonification. Isolation of proteins was started by centrifugation (10,000 g, 4 C, 15 min) and the supernatant was loaded onto a nickel-nitrilotriacetic acid agarose column (Qiagen). Before protein elution with buffer (50 mM sodium phosphate, pH 7.6, 300 mM sodium chloride) containing 500 mM imidazole, the column was washed three times with buffer containing increasing imidazole concentrations (20, 50 and 100 mM). After purification, the protein was dialyzed against 20 mM Hepes-KOH, pH 7.6 (HEPES).
Purification of IM30 E. coli cells were broken by ultrasonification. Isolation of proteins was started by centrifugation (10,000 g, 4 C, 15 min) and the supernatant was loaded onto a nickel-nitrilotriacetic acid agarose column (Qiagen). Before protein elution with buffer (50 mM sodium phosphate, pH 7.6, 300 mM sodium chloride) containing 500 mM imidazole, the column was washed three times with buffer containing increasing imidazole concentrations (20, 50 and 100 mM). After purification, the protein was dialyzed against 20 mM Hepes-KOH, pH 7.6 (HEPES).
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