INMAP was cloned into the pET30a (+) vector (Invitrogen, USA). The resulting construct was transformed into the bacterial strain BL21 (TransGen Biotech, China) for expression. His-tagged protein was purified on Ni2+ beads (GE, USA) from BL21 lysates. For His Pulldown assays, Ni2+ beads with INMAP were incubated with HeLa cell extract for 2 h, isolated by centrifugation, washed, and eluted by boiling in Laemmli sample buffer.
Ni2 beads
Manufactured by GE Healthcare
Sourced in United States
Ni2+-beads are a laboratory product used for protein purification. They consist of nickel-charged agarose beads that can selectively bind to proteins containing a histidine tag, allowing for the isolation and purification of these tagged proteins from complex mixtures. The core function of Ni2+-beads is to facilitate the affinity-based separation and concentration of target proteins.
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4 protocols using ni2 beads
1
Purification and Pulldown of INMAP
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INMAP was cloned into the pET30a (+) vector (Invitrogen, USA). The resulting construct was transformed into the bacterial strain BL21 (TransGen Biotech, China) for expression. His-tagged protein was purified on Ni2+ beads (GE, USA) from BL21 lysates. For His Pulldown assays, Ni2+ beads with INMAP were incubated with HeLa cell extract for 2 h, isolated by centrifugation, washed, and eluted by boiling in Laemmli sample buffer.
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INMAP was cloned into the pET30a (+) vector (Invitrogen, USA). The resulting construct was transformed into the bacterial strain BL21 (TransGen Biotech, China) for expression. His-tagged protein was purified on Ni2+ beads (GE, USA) from BL21 lysates. For His Pulldown assays, Ni2+ beads with INMAP were incubated with HeLa cell extract for 2 h, isolated by centrifugation, washed, and eluted by boiling in Laemmli sample buffer.
2
Purification and Kinase Assays for Rad53, Crt1, and Mck1
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To expressed His6-Rad53 and His6-rad53-KD, the pET-15b-RAD53 (a kind gift from Dr. John Diffley) and pET-15b-rad53-KD (K227A) plasmids were introduced in BL21-codon-plus (DE3)-RIL E. coli strain (Stratagene). His6-Crt1-(201–453) or His6-Crt1-(203–453)-S299D were cloned into a pET28a plasmid. Early log phase culture was treated with 0.2 mM IPTG to induce protein expression. After 3 h of incubation at 25°C, cells were harvested. The proteins were purified using Ni2+-beads (GE Healthcare) and eluted by 250 mM imidazole. pRS-313-pADH1-MCK1-5FLAG plasmid was transformed into an mck1Δ strain. Mck1-5FLAG was purified by 20 μl anti-FLAG M2 beads (Sigma) and eluted by 150 μl of 1 μg/μl FLAG peptide. In a typical kinase assay, 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20, 10 mM MgCl2, 5 μCi of ɣ-32P-ATP were used. Each kinase reaction contained His6-Rad53 (0–10 μg) or His6-Rad53-KD (10 μg) and Mck1-5FLAG (10 μl), or His6-Crt1-(201–453) (8 μg) in 40 μl reaction volume and incubated for 30 min at 30°C. Kinase assay was stopped by heating at 100°C for 5 min in SDS sample buffer. Samples were then subject to SDS–PAGE. Phosphorylation was detected by 32P autoradiography. The amount of protein loaded was detected by CBB staining.
3
Affinity Purification of Protein Complexes
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293F cells expressing FLAG-tagged MMS22L, GSK3A or GSK3B were washed with PBS twice and lysed in the lysis buffer. Proteins were purified by anti-FLAG M2 beads (Sigma) and washed three times in washing buffer (50 mM Tris–HCl, 500 mM NaCl, 1% [v/v] NP-40, 5% [v/v] glycerin, 2 mM PMSF, 10 mM NaF, 20 mM β-glycerophosphate, 100 μM Na3VO4). Proteins were then eluted in the lysis buffer supplemented with 2 mg/ml FLAG peptide. The eluent components were collected and stored in aliquots at -80°C before use.
FLAG-tagged Mck1 was purified from BY4741 yeast strain. The pRS313-pADH1-MCK1-5FLAG plasmid was transformed into an mck1Δ strain. 200 OD600 units of logarithmic cells were lysed by glass bead beating (Mini-Beadbeater-16, Biospec) in the lysis buffer (50 mM Tris–HCl, pH7.5, 500 mM NaCl, 1% [v/v] NP-40, 10% [v/v] glycerin, 2 mM PMSF, 10 mM NaF, 20 mM β-glycerophosphate, 100 μM Na3VO4). Mck1 was purified by anti-FLAG M2 beads (Sigma) and eluted by 1 mg/ml FLAG peptide.
BL21-codon-plus (DE3)-RIL E. coli strain (Stratagene) was transformed with pET vectors expressing His × 6-FLAG-Mms22N or His × 6-Eco1. The expression was induced at OD600 = 0.6 with 0.02 mM IPTG at 25°C for 3 h. The protein was purified using Ni2+-beads (GE Healthcare) and eluted with 500 mM imidazole.
FLAG-tagged Mck1 was purified from BY4741 yeast strain. The pRS313-pADH1-MCK1-5FLAG plasmid was transformed into an mck1Δ strain. 200 OD600 units of logarithmic cells were lysed by glass bead beating (Mini-Beadbeater-16, Biospec) in the lysis buffer (50 mM Tris–HCl, pH7.5, 500 mM NaCl, 1% [v/v] NP-40, 10% [v/v] glycerin, 2 mM PMSF, 10 mM NaF, 20 mM β-glycerophosphate, 100 μM Na3VO4). Mck1 was purified by anti-FLAG M2 beads (Sigma) and eluted by 1 mg/ml FLAG peptide.
BL21-codon-plus (DE3)-RIL E. coli strain (Stratagene) was transformed with pET vectors expressing His × 6-FLAG-Mms22N or His × 6-Eco1. The expression was induced at OD600 = 0.6 with 0.02 mM IPTG at 25°C for 3 h. The protein was purified using Ni2+-beads (GE Healthcare) and eluted with 500 mM imidazole.
4
Protein Domain Expression and Purification
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Protein domains (Table 5) were expressed in E.coli BL21-Gold (DE3), grown in 2YT (10 g/L yeast extract, 16 g/L tryptone and 5 g/L NaCl) and induced at OD600 = 0.7 with 1 mM isopropyl b-D-1-thiogalatactopyranoside followed by 16 h incubation at 18 °C. All domains were glutathione-S-transferase (GST)-tagged, except for the PDZ domains, which were expressed with a maltose binding protein (MBP)-tag. The GST-tagged constructs were purified with glutathione beads (GE Healthcare), whereas ion metal affinity chromatography using Ni 2+ beads (GE Healthcare) was used for PDZ domains. For affinity measurements, the GST tag was removed by thrombin or tobacco etch virus protease (SNX17 FERM) cleavage, whereas the fluorescence polarisation experiments of the PDZ domains were performed on the tagged protein domains. After purification, protein domains were dialysed in 50 mM sodium phosphate buffer pH 7.4 1 mM DTT or PBS pH 7.4 3 mM DTT (PDZ domains). For the complex in displacement experiments, proteins were supplemented with 0.05 % Tween.
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