His-tagged G5845–1508 (His-G5845–1508) (4 pmol) were coupled to Ni-agarose resin (25 μl of beads suspension) (QIAGEN) during 1 h, at 4°C in binding buffer (RBB) (50 mM TrisOAc, pH 7.7, 50 mM KOAc, 5 mM Mg (OAc)2, 10 mM DTT, and 30 μg/ml tRNA). Unbound protein was removed by three washes with RBB, spinning at 14,000g 3 min at 4°C. Beads–protein complexes, resuspended in 100 μl of RBB, were incubated with 80S ribosomes (0.7 pmol) during 1 h, at 4°C. After three washes of the beads complexes with RBB supplemented with NP40 0.05%, spinning at 14,000g 3 min at 4°C, beads-bound proteins were dissolved in SDS-loading buffer, heated at 92°C 3 min, resolved by SDS–PAGE, and detected by WB.
Ni agarose resin
Manufactured by Qiagen
Sourced in Germany
Ni-agarose resin is a chromatographic matrix used for the purification of histidine-tagged recombinant proteins. It consists of nickel-charged agarose beads that selectively bind to the histidine residues on the target proteins, allowing their separation and capture from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Pierce glutathione magnetic beads, by Thermo Fisher Scientific (2 mentions)
Magnegst protein purification system, by Promega (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Superdex 200, by GE Healthcare (1 mentions)
Avitag, by Avidity (1 mentions)
6 ml resource q column, by GE Healthcare (1 mentions)
Akta pure system, by GE Healthcare (1 mentions)
10 protocols using ni agarose resin
1
Ribosome Binding Assay with His-G5
2
Recombinant Superantigen Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genes encoding high-affinity Vβ proteins (Vβ-SEA, Vβ-SEB, Vβ-TSST-1, Vβ-SpeA and Vβ-SpeC) were cloned with a N-terminal 6X-His tag and a C-terminal Avitag (Avidity, LLC) in pET28a expression vector (Novagen) for protein expression in E.coli BL21 (DE3) cells as inclusion bodies. The proteins were refolded (by slow dilution) from denatured inclusion bodies, followed by affinity purification with Ni agarose resin (Qiagen) and HPLC (Biocad Sprint) using a Superdex 200 (GE Healthcare) size exclusion column as described previously [33 (link)].
3
Expression and Purification of Human Ago2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA for MID-domain of human Ago2 (His-Sumo-tagged) was provided by Bhushan Nagar (McGill University). Human eIF1A and mutants of eIF1A-ND/-CD/-GD were inserted into EcoRI/NotI sites of pGEX-4E-1 vectors with GST fusion at the N-term. Human Ago2 (full length) and fragments of L1(172 ~ 227aa), PAZ(227 ~ 349aa), MID (432 ~ 575aa) and PIWI (590 ~ 816aa) were inserted into NdeI/BamHI sites of a pET-6H2 vector. His-tagged proteins expressed in BL21(DE3) cells were extracted by sonication with lysis buffer (50mM Tris, 350mM NaCl, 10mM Imidazole, pH8.0, 20μM Benzonase nuclease, 10μg/ml RNase A, 100μg/ml lysozyme, 2% NP-40, 0.05% β-mercaptoethanol and protease inhibitor) followed by Ni-agarose resin (Qiagen) purification. GST-tagged proteins expressed in BL21(DE3) cells were extracted by MagneGST™ Protein Purification System (Promega) or Pierce Glutathione Magnetic Beads (Thermo Scientific).
4
Purification of HIV-1 Gag Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CTD-SP1 (residues 281–377 of HIV-1 NL4-3 Gag) was cloned with a non-cleavable His-tag (His6-Gly2) into pET30a (Novagen/EMD Millipore, Germany). The construct also contained the P373T substitution, which is a natural sequence variant (Kuiken et al., 2003 (link)). Protein was expressed in E. coli BL21(DE3) cells by induction with 1 mM IPTG for 4 hr at 25°C in shake cultures. Bacteria were harvested by centrifugation and resuspended in 50 mM Tris, pH 8.3, 1 M LiCl, 10 mM β-mercaptoethanol (βME) supplemented with 0.3% (w/v) deoxycholate and protease inhibitor tablets (Roche). Cells were lysed by incubation with lysozyme and sonication. Lysates were clarified by centrifugation and then incubated with Ni-agarose resin (Qiagen, Germany) for 30 min at 4°C. Bound fractions were washed and eluted with a step gradient of 15–300 mM imidazole. The protein was purified to homogeneity using anion exchange and size exclusion chromatography in 20 mM Tris, pH 8.0, 0.5 M NaCl, 20 mM βME. Pure proteins were concentrated to 15–20 mg/mL.
5
Gemin5 Ribosomal Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
His-tagged Gemin5 (His-G5), His-G51–739 wild type and the mutants W14A, Y15A, F381A and Y474A (a kind gift of Dr Chao Xu and Dr Jinrong Min) or His-G51287-1508 proteins (4 pmols) were coupled to Ni-agarose resin (25 μl of beads suspension) (Qiagen) during 1 h at 4°C in binding buffer (RBB) (50 mM TrisOAc pH 7.7, 50 mM KOAc, 5 mM Mg (OAc)2, 10 mM DTT, 30 μg/ml tRNA). Unbound protein was removed by 3 washings with RBB, spinning at 14 000 g 3 min at 4°C. Beads-protein complexes, resuspended in 100 μl of RBB, were incubated with 80S ribosomes, 40S or 60S ribosomal subunits (0.7 pmol) 1 h at 4°C. After three washes of the beads-complexes with RBB supplemented with NP40 0.05%, spinning at 14 000 g 3 min at 4°C, beads-bound proteins were resuspended in SDS-loading buffer, heated at 92°C 3 min, resolved by SDS-PAGE and detected by WB using anti-His (Gemin5), anti-RACK1 (40S) or 3BH5 (anti-P0, 60S and 80S) antibodies. Independent binding assays were conducted at least three times.
6
Expression and Purification of Human Ago2
7
Structural Characterization of eIF1A-SMT3-MID
8
Structural Characterization of eIF1A-SMT3-MID
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BL21(DE3) bacterial cells harboring His-SMT3, His-SMT3-tagged MID(432 ~ 575aa) and His-tagged eIF1A grown in either LB or minimal media containing 15N Ammonium Chloride. The resulting cell pellets were lysed by sonication or french press with lysis buffer (50mM Tris, 350mM NaCl, 10mM Imidazole, pH8.0, 20μM Benzonase nuclease, 10μg/ml RNase A, 100μg/ml lysozyme, 2% NP-40, 0.05% β-mercaptoethanol, protease inhibitor). The soluble proteins were loaded onto a Ni-agarose resin (Qiagen) and proteins were eluted with Ni-elution buffer (50mM Tris, 350mM NaCl, 350mM Imidazole, pH8.0). Purified proteins in Ni-elution buffer were further purified by size exclusion chromatography using a S75 column and eluting with HEPES buffer (20mM HEPES, 150mM NaCl, 0.5mM TCEP, pH7.2). The HSQC measurements were carried out on a Bruker 500 MHz and Varian 600 MHz instrument each equipped with a cryogenically cooled probe. The data was processed using NMRPipe and visualized using CARA. The molar ratios of 15N-eIF1A: SMT3-MID at 1:1 were analyzed in this study and the concentration of 15N-eIF1A was at 200μM. Similarly 15N-SMT3-MID was held at concentration of 200μM and unlabeled eIF1A was titrated at molar ratio of 1:1.
9
Purification of Recombinant LmxMBA Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain the recombinant protein LmxMBA, E. coli BL21 pLysS cells were transformed with the recombinant plasmid pRSETA::LmxMBA and grown in Luria Bertani medium to an optical density of 0.6 at 540 nm. Cells were induced by incubation with 0.1 mM IPTG at 37°C/2 h. The cells were then harvested by centrifugation, washed in ice-cold 50 mM Tris HCl-buffer (pH 7.5), and suspended in extraction buffer (50 mM Tris HCl-buffer (pH 7.5), 150 mM NaCl, 10 mM MgCl2, 5 mM B-mercaptoethanol, 3 M guanidinium chloride, and 2 M urea). After disruption by sonication, the crude extract was clarified by centrifugation at 30,000 × g for 30 min. The rLmxMBA was expressed as a fusion protein with an N-terminus six-histidine–residue affinity tag and was purified, under denatured conditions by affinity chromatography using Ni-agarose resin (Qiagen, Hilden, DE) according to the manufacturer instructions. The collected protein was dialyzed for 48 h at 4°C against PBS.
10
Purification of Recombinant Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression was carried out as reported previously [12] . For protein purification, cells were resuspended in 50 mM HEPES buffer, 300 mM NaCl, 20 mM imidazole, pH 8.0 (buffer A) to a wet cell concentration of 200 mg/mL. After ultrasonication and ultracentrifugation, immobilized metal ion affinity chromatography (IMAC) was employed as a first purification step using Ni-agarose resin (Qiagen) in a gravity flow column following the manufacturer instructions. Anion-exchange chromatography was carried out using a 6-ml RESOURCE Q column (GE Healthcare) on a € AKTA Pure system (GE Healthcare). Size-exclusion chromatography was performed in 50 mM HEPES, 300 mM NaCl, pH 8 on a € AKTA Pure system equipped with a HiLoad 16/600 Superdex 200 pg column. Enzyme elution was monitored at 280 nm and red fractions displaying at least 50% maximal absorbance of the main peak at 280 nm were analyzed by SDS-PAGE (Fig. S1), collected, concentrated and desalted in 50 mM HEPES pH 8 using a PD-10 column. Prior to crystallization trials, protein solutions were concentrated to 20 mg/mL, as determined by NanoDrop (Thermo Scientific), using ε 280 ¼ 67755 M À1 cm À1 and stored overnight at 4 C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!