Myc/his-tagged WT or S208A/S242A mutant N-terminal pyrin (aa’s 1- 330) was overexpressed into 293T cells, and the N-terminal pyrin proteins were purified using His-SpinTrap™ (28-4013-53, GE Healthcare). Each purified N-terminal pyrin protein was incubated with 5 μg of PKN1 (PR7255B) or PKN2 (PR7370A, Thermo Scientific) at 30°C for 0.5h in a kinase buffer (50 mM HEPES pH7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 500 μM ATP and 2.5 mM DTT). The reactants were analyzed by immunoblotting with anti-phospho-Ser antibody (9606, Cell Signaling) or staining with Pro-Q diamond (P33300, Invitrogen). Pro-Q diamond-stained gel was visualized on Molecular Imager FX (Bio-Rad).
His spintrap
Manufactured by GE Healthcare
Sourced in United States
His SpinTrap is a laboratory centrifuge designed for high-speed separation of biological samples. It is capable of achieving high relative centrifugal forces to efficiently pellet cells, organelles, and macromolecules from complex mixtures. The device features a compact and user-friendly design for convenient benchtop operation.
Automatically generated - may contain errors
Lab products found in correlation
Spintrap, by Cytiva (8 mentions)
Pro q diamond, by Bio-Rad (2 mentions)
Molecular imager fx, by Bio-Rad (2 mentions)
Pro q diamond, by Thermo Fisher Scientific (2 mentions)
Anti phospho ser antibody, by Cell Signaling Technology (2 mentions)
Bio safe coomassie blue, by Bio-Rad (1 mentions)
Taq dna polymerase, by New England Biolabs (1 mentions)
Mini protein tetra system, by Bio-Rad (1 mentions)
His gravitrap, by GE Healthcare (1 mentions)
Gravitrap, by Cytiva (1 mentions)
Anti his g antibody, by Thermo Fisher Scientific (1 mentions)
Pierce chromogenic endotoxin quant kit, by Thermo Fisher Scientific (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Avantor (1 mentions)
Lb medium, by Duchefa Biochemie (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
Pfastbac1, by Thermo Fisher Scientific (1 mentions)
Primestar max dna polymerase premix, by Takara Bio (1 mentions)
Ni sepharose high performance affinity media, by GE Healthcare (1 mentions)
11 protocols using his spintrap
1
Phosphorylation of N-Terminal Pyrin
2
Purification of His-tagged Proteins
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Chemicals reagents were from Sigma-Merck. Deoxynucleosides, dNMPs, dNTPs, AMP, ADP and ATP were all of >99% purity. Acetic anhydride and H3PO4 (85%) used for the synthesis of acetyl phosphate were ACS grade. Taq DNA polymerase and broad range (10–200 kDa) molecular weight markers were from New England Biolabs (MA, USA). SDS-PAGE gels (15%) were run on a Mini-protein Tetra system and stained with Bio-safe Coomassie Blue (Biorad). His-tagged proteins were purified with His-GraviTrap or His-SpinTrap kits from GE Healthcare.
3
Immobilized Artificial Metalloenzyme Catalysis
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His SpinTrap (GE Healthcare,
USA) purification columns were adapted for batch reactions in a thermoshaker
for microtubes (Grant-bio). Prior to first use, the original content
was removed, and the cartridges were washed thoroughly with distilled
water. Then, 50 μL of 250 μM immobilized ArM suspension
was poured into a precleaned His SpinTrap cartridge, and storage solvent
was removed by centrifugation (500×g, 1 min).
The bottom outlet was sealed, 450 μL of catalytic buffer was
added, and the vial was transferred to a shaker for preconditioning
at the desired temperature for 5 min. In sequence, 50 μL of
20 mM 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline (1 ) in catalytic buffer was added, the vial was capped and shaking
immediately started. The pH, shaking, and temperature effects were
investigated. Recyclability and redox-reversible assembly studies
were also carried out in these adapted reactors.Figure 2 b summarizes the adapted setup,
which provided a straightforward way of separating the immobilized
ATHases from the reaction medium and facilitated recycling by centrifugation
(500×g, 1 min). Further details can be found
in the supplementary methods, including the homogeneous catalytic
reactions, the sample preparation for analytical measurements, and
the redox release and recharging of the scaffold for recyclability
tests.
USA) purification columns were adapted for batch reactions in a thermoshaker
for microtubes (Grant-bio). Prior to first use, the original content
was removed, and the cartridges were washed thoroughly with distilled
water. Then, 50 μL of 250 μM immobilized ArM suspension
was poured into a precleaned His SpinTrap cartridge, and storage solvent
was removed by centrifugation (500×g, 1 min).
The bottom outlet was sealed, 450 μL of catalytic buffer was
added, and the vial was transferred to a shaker for preconditioning
at the desired temperature for 5 min. In sequence, 50 μL of
20 mM 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline (
immediately started. The pH, shaking, and temperature effects were
investigated. Recyclability and redox-reversible assembly studies
were also carried out in these adapted reactors.
which provided a straightforward way of separating the immobilized
ATHases from the reaction medium and facilitated recycling by centrifugation
(500×g, 1 min). Further details can be found
in the supplementary methods, including the homogeneous catalytic
reactions, the sample preparation for analytical measurements, and
the redox release and recharging of the scaffold for recyclability
tests.
4
Phosphorylation of N-Terminal Pyrin
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Myc/his-tagged WT or S208A/S242A mutant N-terminal pyrin (aa’s 1- 330) was overexpressed into 293T cells, and the N-terminal pyrin proteins were purified using His-SpinTrap™ (28-4013-53, GE Healthcare). Each purified N-terminal pyrin protein was incubated with 5 μg of PKN1 (PR7255B) or PKN2 (PR7370A, Thermo Scientific) at 30°C for 0.5h in a kinase buffer (50 mM HEPES pH7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 500 μM ATP and 2.5 mM DTT). The reactants were analyzed by immunoblotting with anti-phospho-Ser antibody (9606, Cell Signaling) or staining with Pro-Q diamond (P33300, Invitrogen). Pro-Q diamond-stained gel was visualized on Molecular Imager FX (Bio-Rad).
5
Overexpression and Purification of EstATII
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Example 5
200 ml of E. coli BL21 (DE3) harboring the pET-SUMO/EstATII plasmid were grown in LB at 37° C. until the culture reached an OD600=0.4-0.6. The culture was induced by adding isopropyl-b-D thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and further incubated for 3 hours at 37° C. The cells were harvested by centrifugation at 10,000.times.g for 15 min at 4° C. The purification procedure was performed using the His SPINTRAP™ (GE Healthcare) according to manufacturer's instructions. The purified protein was dialysed against 50 mM NaH2PO4 buffer, pH 8.0, analyzed by SDS-PAGE and stored at 4° C. until further use. Protein isolation was confirmed by Western Blot analysis using an anti-His-G antibody (Invitrogen).
Results: Overexpression and Purification of EstATII
In order to investigate the biochemical characteristics of EstATII, the gene was expressed as an N-terminal His-tag recombinant protein in the expression vector pET-SUMO in E. coli BL21 (DE3) cells. The overexpressed protein had the expected molecular weight of approximately 46 kDa and western blot analysis of the purified protein showed a single band at the expected size (
6
Purification of Recombinant Proteins from E. coli
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The Mdh was prepared according to a previously described method [14 (link)]. Briefly, E. coli strain harboring a recombinant plasmid constructed in a previous study [15 (link)] was grown overnight at 37° C in LB medium (Duchefa, Netherlands) and protein expression was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG, Amresco, USA) at a final concentration of 0.3 mM. Then, the soluble protein fraction was purified using a His SpinTrap (GE Healthcare, UK) column according to the manufacturer's instructions after the pellet was sonicated at 10,000 Hz on ice. The proteins were constructed with a pCold vector containing a chaperone trigger factor (TF) to produce soluble and functional proteins. Therefore, as a vector control, TF was also purified using the above method. The endotoxin content of proteins was confirmed with a Pierce Chromogenic Endotoxin Quant kit (ThermoFisher, USA). The purity of proteins was confirmed using SDS-PAGE and Western blotting as previously described [15 (link)]. The concentration of purified proteins was measured by Pierce BCA protein assay kit (Pierce, USA) following the manufacturer’s instructions and stored at −20°C until use.
7
EphA2-specific scFv Antibody Production
8
Recombinant BmIDGF Production and Rescue Assay
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The recombinant BmIDGF was produced using a Bac-to-Bac system (Invitrogen) as described previously [49 (link)]. The primers used to construct the vectors are listed in S2 Table
9
Cloning and Purification of Phosphomevalonate Kinase
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The ERG8 gene (NM_001182727.1) encoding the phosphomevalonate kinase in Saccharomyces cerevisiae was amplified by PCR containing PrimeSTAR MAX DNA polymerase premix (Takara Bio, Inc.) using the primers 5′-TCAGAGTTGAGAGCCTTCAGTGCCCCAG-3′ and 5′-GGAATTC TCTTTATCAAGATAAGTTTCCGGATCTTTTT-3′ and genomic DNA from S. cerevisiae as a template. pET21-d(+) was digested with NcoI, treated with the Klenow fragment of DNA polymerase I, and then digested with EcoRI. The PCR-generated DNA fragment was digested with EcoRI and ligated with the described vector fragment. The expected DNA sequence of the inserted fragment was confirmed by sequencing.
E. coli BL21(DE3) was transformed with the obtained plasmid and cultured in 20 ml of LB broth at 30 °C by reciprocal shaking at 140 r.p.m. When the OD600 reached approximately 0.7, 0.1 mM IPTG was added and cultivation was continued overnight under the same conditions. Cells were harvested by centrifugation, resuspended in buffer solution A (50 mM sodium phosphate, 0.3 M NaCl and 20 mM imidazole) and disrupted by ultrasonication. After centrifugation, the resulting supernatant was adsorbed onto a His SpinTrap (GE Healthcare) column and the adsorbed proteins were eluted with eluting solution (buffer solution A containing 0.5 M imidazole). The obtained eluate was dialyzed with 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl as the external solution.
E. coli BL21(DE3) was transformed with the obtained plasmid and cultured in 20 ml of LB broth at 30 °C by reciprocal shaking at 140 r.p.m. When the OD600 reached approximately 0.7, 0.1 mM IPTG was added and cultivation was continued overnight under the same conditions. Cells were harvested by centrifugation, resuspended in buffer solution A (50 mM sodium phosphate, 0.3 M NaCl and 20 mM imidazole) and disrupted by ultrasonication. After centrifugation, the resulting supernatant was adsorbed onto a His SpinTrap (GE Healthcare) column and the adsorbed proteins were eluted with eluting solution (buffer solution A containing 0.5 M imidazole). The obtained eluate was dialyzed with 20 mM Tris-HCl (pH 8.0) containing 50 mM NaCl as the external solution.
10
Purification of His-tagged Proteins
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All purification steps were performed at room temperature as described previously [17 (link)]. His-tag affinity spin columns (His SpinTrap; GE Healthcare BioSciences, Pittsburgh, PA, USA) were used to purify the protein. The column was first equilibrated with binding buffer (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.5). Cell extracts (in 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.5) were applied to the column, and then the column was washed twice with wash buffer (50 mM sodium phosphate, 500 mM NaCl, 50 mM imidazole, 20% ethanol, pH 7.5). The His-tagged protein was eluted with elution buffer (50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.5).
For large volume purification, Ni Sepharose high performance affinity media (GE Healthcare BioSciences, Pittsburgh, PA, USA) and Glass Econo-Column® Columns 2.5 × 10 cm (BioRad, Hercules, CA, USA) were used.
For large volume purification, Ni Sepharose high performance affinity media (GE Healthcare BioSciences, Pittsburgh, PA, USA) and Glass Econo-Column® Columns 2.5 × 10 cm (BioRad, Hercules, CA, USA) were used.
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