Recombinant CPAF constructs were generated by cloning C. trachomatis CPAF into either pET28b (C-terminal His-tagged) or pETM11 (N-terminal GST-tagged) constructs. Similarly, human PI15 was cloned into pET28b vector (C- as well as N-terminal His-tagged). TEV protease cut sites are introduced between PI15 ORF and His coding sequences. For recombinant protein expression, protein-coding constructs were transformed into E. coli pRARE strain and were grown overnight at 15°C in presence of 0.5 M IPTG. Bacterial cells were pelleted down, lysed with 50 mM Hepes pH = 7.5 containing 100 mM NaCl, 5 mM β-Mercaptoethanol, 0.5 mM PMSF and necessary protease inhibitors. After removal of cell debris, soluble recombinant proteins were eluted by either Glutathione beads or Ni-NTA beads (Thermo Fischer) wherever necessary. Purified His-PI15-His proteins were digested with TEV protease and untagged PI15 proteins were eluted out. Eluted proteins were further purified by gel-filtration chromatography using Supadex 200 and sepharose 6 columns.
Ni nta beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Ni-NTA beads are a type of agarose beads coated with nickel ions (Ni2+). They are used for the purification of proteins with a histidine-tag (His-tag) through affinity chromatography. The His-tag binds to the Ni2+ ions on the beads, allowing the target protein to be separated from other components in the sample.
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Lab products found in correlation
Monoclonal antibody d 3, by Santa Cruz Biotechnology (2 mentions)
His tagged protein purification binding buffer, by Thermo Fisher Scientific (2 mentions)
Glutathione beads, by Thermo Fisher Scientific (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Superdex 75 10 300 gl column, by GE Healthcare (1 mentions)
Complete edta free protease inhibitor cocktail, by Roche (1 mentions)
Instantblue, by Abcam (1 mentions)
E coli total lipids, by Avanti Polar Lipids (1 mentions)
Ni nta, by Thermo Fisher Scientific (1 mentions)
Anti human cd3 apc, by BioLegend (1 mentions)
Akta fplc, by GE Healthcare (1 mentions)
Infinite m200, by Tecan (1 mentions)
Usb2000 spectrophotometer, by OceanOptics (1 mentions)
Sybr green master mix, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Zeba desalt spin column, by Thermo Fisher Scientific (1 mentions)
Streptavidin sepharose bead, by GE Healthcare (1 mentions)
40 protocols using ni nta beads
1
Recombinant PI15 and CPAF Production
2
Identification of N-cadherin Interactors
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N-cadherin–His protein was incubated with magnetic Ni-NTA beads (88831; Thermo Fisher Scientific) at a concentration of 1 µg/µl for 1 h at room temperature. N-cadherin–His protein was cross-linked to beads using EDC/NHS for 45 min at room temperature (Chevalier et al., 2010 (link)). Beads were incubated with the culture of ECs for 1 h. Proteins were cross-linked with the reversible cross-linker 50 µM DTSP. Beads were then collected from cell lysates using a magnetic separator, washed five times in PBS, and boiled in sample buffer containing 1 mM DTT to break the cross-link. Lysates were separated by SDS-PAGE and silver-stained to highlight the bands of interest. Each lane of the gel was cut into three pieces and submitted to the Taplin Biological Mass Spectrometry Facility, Harvard University, Boston, MA, for analysis by microcapillary liquid chromatography–tandem mass spectrometry.
3
SARS-CoV-2 NP SUMOylation and Ubiquitination
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Cells transfected with the indicated plasmids, or infected with SRAS-CoV-2 (m.o.i. of 0.1) were treated with 5 mM MG132 (SelleckChem, E2899) for 4 h prior to harvesting. After 36-48 h, the cells were washed with PBS containing 10 mM N-ethylmaleimide (NEM, Sigma, 128530) and lysed in two pellet volumes of RIPA buffer supplemented with protease inhibitor cocktail and 10 mM NEM. To detect SARS2-NP SUMOylation in the lungs of SRAS-CoV-2-infected hACE2-transgenic mice, 200 mg of lung was homogenized in 1000 µl of buffer 24 h after infection.
Under denaturing conditions, lysates were sonicated, boiled at 100 °C for 5 min, diluted with RIPA buffer containing 0.1% SDS, and centrifuged at 15,000 g for 15 min at 4 °C. Equal amounts of the lysate supernatants were incubated with anti-NP antibody (3 h) and protein G (1 h) orderly at 4 °C for detecting NP SUMOylation in SRAS-CoV-2-infected cells, or with anti-Flag beads for ubiquitination assay for 3 h at 4 °C. After extensive washing, the bound proteins were eluted with SDS sample buffer and subjected to IB analysis. For the Ni-NTA pulldown of 6His-tagged SUMOylated proteins, cells were resuspended in above lysis buffer containing 8 M urea, and the 6His-tagged proteins recovered with Ni-NTA beads (Thermo Fisher, R90101) were eluted with a buffer containing 8 M urea and 20 mM imidazole.
Under denaturing conditions, lysates were sonicated, boiled at 100 °C for 5 min, diluted with RIPA buffer containing 0.1% SDS, and centrifuged at 15,000 g for 15 min at 4 °C. Equal amounts of the lysate supernatants were incubated with anti-NP antibody (3 h) and protein G (1 h) orderly at 4 °C for detecting NP SUMOylation in SRAS-CoV-2-infected cells, or with anti-Flag beads for ubiquitination assay for 3 h at 4 °C. After extensive washing, the bound proteins were eluted with SDS sample buffer and subjected to IB analysis. For the Ni-NTA pulldown of 6His-tagged SUMOylated proteins, cells were resuspended in above lysis buffer containing 8 M urea, and the 6His-tagged proteins recovered with Ni-NTA beads (Thermo Fisher, R90101) were eluted with a buffer containing 8 M urea and 20 mM imidazole.
4
Purification of α-FLT3 scFv
5
Characterization of PPARγ Protein Interactions
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Human PD‐L1 or LC3B cDNA was cloned into pET28a vector. PPARγ cDNA was cloned into PGEX‐6P. PGEX‐6P‐F70A or PGEX‐6P‐ΔLBD was mutated by the site‐directed mutagenesis method and identified by DNA sequencing. GST‐PPARγ, GST‐F70A, GST‐ΔLBD, his‐LC3B, and his‐PD‐L1 were expressed in E. coli strain BL21(DE3) (pAPlacIQ). The recombinant proteins were purified using glutathione beads or Ni‐NTA beads (Thermo Fisher). For in vitro binding of PPARγ to PD‐L1, GST‐PPARγ or GST‐ΔLBD (5 μg) fusion protein was immobilized on glutathione‐agarose beads in buffer (25 mM HEPES pH 7.5, 0.2% NP‐40, 6 mM NaCl) for 1 h at 4°C, then the same amount of recombinant his‐PD‐L1 was added. For in vitro binding of PPARγ to LC3, GST‐PPARγ or GST‐F70A (5 μg) fusion protein was immobilized on glutathione‐agarose beads in buffer (25 mM HEPES pH 7.5, 0.2% NP‐40, 6 mM NaCl) for 1 h at 4°C, then the same amount of recombinant his‐LC3B was added. These reactions were incubated for another 1 h. Adsorbates to glutathione‐conjugated beads were analyzed by Western blot.
6
Purification of His-Tagged Proteins
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Bacterial cell pellets
were thawed, resuspended in lysis buffer (50 mM HEPES, pH 6.8, 1 M
NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol) containing complete
EDTA-free protease inhibitor cocktail (Roche; Mannheim, Germany),
and lysed for a total of 3 min of sonication at 50% power using a
Branson Sonifier. Lysates were clarified by centrifugation at 13 000
rpm (20,064g) for 20 min in an F21S-8x50y rotor (Thermo
Fisher Scientific) at 37 °C and incubated with 0.5 mL of Ni–NTA
beads (Thermo Fisher Scientific) at room temperature for 1 h. Beads
were then washed three times with 10 mL of lysis buffer. Proteins
were eluted by addition of lysis buffer containing 500 mM imidazole
and 1 mM DTT. Elutions were diluted to 3 mg/mL in lysis buffer containing
1 mM DTT and dialyzed overnight into single RGG storage buffer (500
mM NaCl, 20 mM HEPES, pH 6.8, 1 mM DTT) using 10 kDa cutoff Slide-A-Lyzer
membrane cassettes (Thermo Fisher Scientific). Proteins were concentrated
by centrifugation in 4 mL of Amicon filter concentrators with a 10
kDa cutoff (Millipore Sigma; Burlington, MA). TCEP (1 mM) was added
prior to snap freezing and storage at −80 °C
were thawed, resuspended in lysis buffer (50 mM HEPES, pH 6.8, 1 M
NaCl, 20 mM imidazole, 1 mM β-mercaptoethanol) containing complete
EDTA-free protease inhibitor cocktail (Roche; Mannheim, Germany),
and lysed for a total of 3 min of sonication at 50% power using a
Branson Sonifier. Lysates were clarified by centrifugation at 13 000
rpm (20,064g) for 20 min in an F21S-8x50y rotor (Thermo
Fisher Scientific) at 37 °C and incubated with 0.5 mL of Ni–NTA
beads (Thermo Fisher Scientific) at room temperature for 1 h. Beads
were then washed three times with 10 mL of lysis buffer. Proteins
were eluted by addition of lysis buffer containing 500 mM imidazole
and 1 mM DTT. Elutions were diluted to 3 mg/mL in lysis buffer containing
1 mM DTT and dialyzed overnight into single RGG storage buffer (500
mM NaCl, 20 mM HEPES, pH 6.8, 1 mM DTT) using 10 kDa cutoff Slide-A-Lyzer
membrane cassettes (Thermo Fisher Scientific). Proteins were concentrated
by centrifugation in 4 mL of Amicon filter concentrators with a 10
kDa cutoff (Millipore Sigma; Burlington, MA). TCEP (1 mM) was added
prior to snap freezing and storage at −80 °C
7
Purification of Active Recombinant PKA Variants
8
Reconstitution and Purification of MsbA
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MsbA was expressed, purified and reconstituted in liposomes as described6 (link), 30 (link). For reconstitution, purified MsbA in 0.065% DDM and 0.04% Na cholate was mixed at 1:10 protein:lipid ratio (w/w) with E. coli total lipids (Avanti Polar Lipids) in 100 mM NaCl, 20 mM Tris/HCl, pH 7.4, with 0.1 mM tris(2-carboxyethyl)phosphine (TCEP). The liposomes containing MsbA were extruded through a 200-nm polycarbonate filter and incubated with SMA or zSMA1 at a final concentration of 2.5% (w/v) for 2 h at room temperature. After incubation, non-solubilized material was removed by centrifugation at 100,000 g for 30 min. The MsbA-loaded nanodiscs were enriched based on the affinity of the His-tagged MsbA for Ni2+. The supernatant was mixed with Ni-NTA beads (Thermo Fisher Scientific) at a ratio of 100 μl of resin/ml of solubilized protein, and incubated at 4 °C overnight with gentle rotation. Then, the samples were transferred to a gravity flow column and the resin was washed with 10 column volumes of 100 mM NaCl, 20 mM Tris/HCl, pH 7.4, with 0.1 mM TCEP and 20 mM imidazole, and elution was achieved by increasing the concentration of imidazole to 200 mM. Eluted fractions were analyzed on gels (16% SDS-PAGE) stained with Instant Blue (Expedeon). The ATPase activity of MsbA was measured using a variant of the ATPase linked assay30 (link), 31 (link).
9
Ni-NTA Pulldown of Rev-A IN with BRD4
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For in vitro Ni-NTA pull-down assays, 10 μg of His6-tagged Rev-A IN in 100 μL pull-down buffer (25 mM Tris HCl pH 7.5, 150 mM NaCl, 25 μM ZnCl2, 0.1% (v/v) Nonidet P40, 20 mM imidazole) was mixed with 10 μL settled volume of Ni-NTA beads (Thermo Scientific) previously washed with pull-down buffer. Following incubation at 4°C for 2 h with gentle agitation, 10 μg BSA and 10 μg of BRD4462–720 or BRD4462–720/L630E were added, and mixtures were incubated overnight at 4°C. The beads were washed five times with pull-down buffer and briefly centrifuged for 1 min at 1,300 g, and were then resuspended in 20 μL 2X sodium dodecyl sulfate (SDS) gel loading buffer and boiled for 10 min. The resulting supernatant was analyzed by denaturing gel electrophoresis on a 10% acrylamide gel. Proteins were detected by staining with Coomassie blue.
10
Recombinant Expression of Mouse and Beaver SIRT6
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