Bacterial expression vectors were transformed into E coli BL21 (DE3) individually. Expression of Rab5a, Diaph1 (1-453), or full-length Diaph1 (Diaph1 FL) was induced by 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) and culture of cells at 16°C, while the expression of TβRII was induced by 50 μM IPTG and culture of cells at 15°C. A lysis buffer, containing 25 mM Tris-HCl pH7.6, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1mg/mL lysosome, 1% NP-40, 1% TritonX-100, was then added to lyse the cells. Cells were incubated on ice for 20 minutes and subjected to sonication until the lysates turned clear. After centrifugation at 15 000 rpm for 15 minutes, the supernatants were collected and GST-fused proteins were purified by Glutathione Sepharose 4B beads (17075601; GE Healthcare). Removal of the GST tag from GST-TβRII fusion protein was performed by PreScission Protease (GE27-0843-01; Millipore Sigma) and removal of the GST tag from GST-Rab5a was performed by Thrombin (10602400001; Millipore Sigma).
Prescission protease
Manufactured by Merck Group
Sourced in United States, Germany
PreScission protease is a lab equipment product that serves as a specific enzyme for the cleavage of fusion proteins. It is a recombinant form of the human rhinovirus 3C protease, which can be used to remove affinity tags from recombinant proteins during purification processes.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b beads, by GE Healthcare (6 mentions)
Gst affinity agarose, by GE Healthcare (3 mentions)
Thrombin, by Merck Group (2 mentions)
Glutathione resin, by GE Healthcare (2 mentions)
Biomol green, by Enzo Life Sciences (1 mentions)
Coomassie staining, by Merck Group (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
P3xflag cmv 10 vector, by Merck Group (1 mentions)
Glutathione beads, by GenScript (1 mentions)
Amylose beads, by New England Biolabs (1 mentions)
Superdex 200 16 60 gel filtration column, by GE Healthcare (1 mentions)
Amp pnp, by Merck Group (1 mentions)
Vivaspin 20, by Sartorius (1 mentions)
Igg sepharose beads, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Avance 3 hd 700 mhz, by Bruker (1 mentions)
Pgex6p 2, by Thermo Fisher Scientific (1 mentions)
Xhoi, by Vazyme (1 mentions)
Bamhi, by Vazyme (1 mentions)
600 mhz spectrometer, by Agilent Technologies (1 mentions)
22 protocols using prescission protease
1
Recombinant Protein Expression and Purification
2
Recombinant Protein Expression and Purification
3
Quantifying VCP ATPase Activity
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For ATPase activity of VCP 500 ng of recombinant active GST-VCP (SignalChem, #VCP-195H) was incubated in 50 µL of reaction buffer (10 mM HEPES-KOH pH 7.7, 2.5 mM MgCl2, 50 mM KCl, and 1 mM DTT) together with DMSO, 20 µM SMER28 or DBeQ, NMS873, CB-5083. The reaction was started by the addition of 0,1 mM ATP and carried out for 1 h at room temperature. The reaction was stopped by the addition of 100 µL BIOMOL green (Enzo LifeSciences, #BML-AK111-0250), and after 30 min absorbance at 650 nm was determined and the amount of released phosphate was interpolated from a standard curve. For ATPase activity of WT VCP, ATPase mutants and the VCP-R155H mutant, GST-tagged VCP proteins were purified from E. coli (GST-tag removed by PreScission protease; Merck, #GE27-0843-01), and used in ATPase assay buffer (20 mM HEPES-KOH pH 7.7, 20 mM MgCl2, 50 mM KCl, and 1 mM DTT). Reaction was started by the addition of 2 mM ATP and carried out at 37 °C for denoted time points, followed by the addition of 100 µL BIOMOL green and absorbance reading. 200–500 ng of purified protein was used per reaction.
4
Purification of Recombinant Yeast Polη
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Human Polη was overexpressed as N-terminal fusion with glutathione S-transferase (GST) in Saccharomyces cerevisiae BJ5464 protease deficient strain, and affinity purified on glutathione–Sepharose 4B beads (GE Healthcare, Uppsala, Sweden) using the same protocol as for yeast Polη [28 (link)]. The GST-tag was removed in the last step of the purification by incubating the beads with PreScission protease (Merck KGaA, Darmstadt, Germany). The efficiency of the purification was verified by polyacrylamide gel electrophoresis and Coomassie staining (Merck KGaA, Darmstadt, Germany), and the protein concentration was determined using a Nanodrop spectrophotometer and a gel-based assay.
5
Purification of Saccharomyces cerevisiae Polη
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Saccharomyces cerevisiae Polη was overexpressed in yeast in N-terminal fusion with GST and affinity purified on glutathione–Sepharose beads (GE Healthcare, Uppsala Sweden). as described previously [21 (link)]. The GST-tag was removed in the last step of the purification by incubating the beads with PreScission protease (Merck KGaA, Darmstadt, Germany). Efficiency of the purification was verified by polyacrylamide gel electrophoresis and Coomassie staining (Merck KGaA, Darmstadt, Germany).
6
Purification and Characterization of MPP1 Mutants
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All the sequences of primers for the construction of plasmids used in this study are summarized in the Supplementary Table 1 . The MPP1-truncated mutants (MPP1-Mut1-5) were subcloned into the pGEX-6p1 vector and expressed as soluble GST tagged proteins in Escherichia coli BL21 (DE3) or LEMO (DE) cells. Full length MPP1-GST and its truncated mutants were isolated in native conditions based on HBS buffers (10 mM HEPES, 150 mM NaCl, pH 7,4) and immobilized on glutathione-Sepharose 4B beads (GE Healthcare). The GST tag was cleaved off on the column with the PreScission protease (Sigma) according to the manufacturer's protocol. Purified recombinant proteins were validated by SDS-PAGE and Coomassie Blue staining (see Fig. S2 ). His-tagged flotillin 1 and 2 were purified under denaturation conditions as described previously23 (link). After purification recombinant proteins were dialyzed into HBS-T (HBS-0.05% Tween-20) buffer and subsequently used for SPR binding study. For mammalian cell experiments the MPP1-Mut4-FLAG construct was additionally cloned into the p3XFLAG-CMV-10 vector (Sigma, St. Louis, MO).
7
Recombinant Tau Protein Purification
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The longest human tau isoform tau441 and its truncation form, tau151–391, were constructed into pGEX‐6p‐1 and expressed in BL21‐Gold E. Coli with 0.5 mM IPTG induction for 3 h at RT. The bacteria were lysed by sonication in 50 mM Tris–HCl, pH 7.4, 0.15 M NaCl, 1 mM dithiothreitol (DTT), 1.0 mM Na3VO4, 50 mM NaF, 1.0 mM AEBSF, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin. Triton X‐100 was added to bacterial lysates to 1% final concentration and centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was incubated with glutathione agarose beads for 1 h at 4°C. After extensive washing, the glutathione agarose beads were incubated with PreScission™Protease (Sigma, St. Louis, MO, USA) in buffer (50 mM Tris–HCl, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) for 16 h at 4°C. Cleaved recombinant tau (rTau) eluted from beads was collected, dialyzed against 10 mM Tris‐buffer (pH 7.4) with 0.5 mM β‐ME, lyophilized, and stored at −80°C until used.
8
Purification and Characterization of Terminase Complexes
9
Affinity Purification of Plant Proteins
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Plants for affinity purification assay were grown in 8 cm3 pots filled with soil S-Jord (Hasselfors) under long day conditions: 150 µE m−2 s−1 light for 16 h, 8 h dark, 22 °C, 70% humidity.
Plant rosette leaves were harvested at bolting stage and flash frozen in liquid nitrogen. Sample weight was >15 g of frozen tissue. Subsequent procedure was performed as previously described [18 (link)] using the following consumables: Protease inhibitor cocktail (P9599-5ML, Sigma-Aldrich, Saint Louis, MO, USA), PreScission protease (GE27-0843-01, Sigma-Aldrich), Ig-G sepharose beads (GE17-0969-01, Sigma-Aldrich),), Ni-sepharose beads (GE17-5318-01, Sigma-Aldrich).
Mass spectrometry data was acquired using a data-dependent acquisition procedure with a cyclic series of a full scan from 350–1500 with resolution of 120,000 control (AGC) target 1E6, maximum injection time 100 ms. The top S (3 s) and dynamic exclusion of 30 s were used for selection of Parent ions for MSMS in the HCD cell with, relative collision energy 30% and scanned in the orbitrap with resolution of 30,000.
Plant rosette leaves were harvested at bolting stage and flash frozen in liquid nitrogen. Sample weight was >15 g of frozen tissue. Subsequent procedure was performed as previously described [18 (link)] using the following consumables: Protease inhibitor cocktail (P9599-5ML, Sigma-Aldrich, Saint Louis, MO, USA), PreScission protease (GE27-0843-01, Sigma-Aldrich), Ig-G sepharose beads (GE17-0969-01, Sigma-Aldrich),), Ni-sepharose beads (GE17-5318-01, Sigma-Aldrich).
Mass spectrometry data was acquired using a data-dependent acquisition procedure with a cyclic series of a full scan from 350–1500 with resolution of 120,000 control (AGC) target 1E6, maximum injection time 100 ms. The top S (3 s) and dynamic exclusion of 30 s were used for selection of Parent ions for MSMS in the HCD cell with, relative collision energy 30% and scanned in the orbitrap with resolution of 30,000.
10
TEAD1 protein binding analysis by STD NMR
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Binding of compounds to human TEAD was performed
using STD NMR analysis. Briefly, TEAD1 protein (residues 194–411)
was prepared by solution cleavage of the GST tag from recombinant
GST–TEAD1 (194–411). GST–TEAD1 protein was eluted
from glutathione beads by 40 mM reduced glutathione. GST tag was removed
by incubation with PreScission protease (Sigma-Aldrich) at 4 °C
for 18 h. Glutathione was removed by dialysis against 1000 volumes
of 1× PBS at 4 °C for 4 h. Free GST protein was removed
by incubation with glutathione resin. All NMR spectroscopy experiments
were performed on a Bruker Avance III HD 700 MHz spectrometer equipped
with a 1.7 mm inverse triple-resonance microcryocoil probe. NMR samples
were prepared in 40 μL with PBS pH 7.4 in 60% D2O
(uncorrected for D2O). TEAD (20 mM) was used with a final
concentration of 2 mM compound. For the STD experiments, the standard
Bruker stddiffesgp.3 pulse sequence was used with a saturation time
of 7 s and a spectral width of 15.9 ppm with eight scans. The on-resonance
frequency was set to 0.85 ppm, while the off-resonance frequency was
set to −28 ppm. Appropriate blank experiments, in the absence
of protein or ligand, were performed to test the lack of direct saturation
to the ligand protons.
using STD NMR analysis. Briefly, TEAD1 protein (residues 194–411)
was prepared by solution cleavage of the GST tag from recombinant
GST–TEAD1 (194–411). GST–TEAD1 protein was eluted
from glutathione beads by 40 mM reduced glutathione. GST tag was removed
by incubation with PreScission protease (Sigma-Aldrich) at 4 °C
for 18 h. Glutathione was removed by dialysis against 1000 volumes
of 1× PBS at 4 °C for 4 h. Free GST protein was removed
by incubation with glutathione resin. All NMR spectroscopy experiments
were performed on a Bruker Avance III HD 700 MHz spectrometer equipped
with a 1.7 mm inverse triple-resonance microcryocoil probe. NMR samples
were prepared in 40 μL with PBS pH 7.4 in 60% D2O
(uncorrected for D2O). TEAD (20 mM) was used with a final
concentration of 2 mM compound. For the STD experiments, the standard
Bruker stddiffesgp.3 pulse sequence was used with a saturation time
of 7 s and a spectral width of 15.9 ppm with eight scans. The on-resonance
frequency was set to 0.85 ppm, while the off-resonance frequency was
set to −28 ppm. Appropriate blank experiments, in the absence
of protein or ligand, were performed to test the lack of direct saturation
to the ligand protons.
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