To obtain Zn(II)-loaded MT proteins aliquots of apo-MT forms in diluted HCl (pH 2.5 with 1 mM TCEP) under a nitrogen blanket were mixed with ZnSO4 depending on previously reported stoichiometries of zinc forms with 1.2–1.5 eq. excess.81 The pH of such solutions was adjusted to 8.6 with a 1 M solution of Tris base.48 (link) MTs were concentrated with Amicon Ultra-4 Centrifugal Filter Units, containing a membrane cut-off of 3 kDa (Merck), and purified on Superdex 75 Increase 10/300 (Cytiva) in 20 mM Tris-HCl, pH 8.6. The concentrations of thiolates were determined using DTNB.54 (link) Zn(II) concentration was analysed using PAR assay and ICP-OES iCAP 7400 Duo (Thermo Scientific). The samples were diluted in 10% HNO3 to confirm spectroscopic measurements.58 (link)
Superdex 75 increase 10 300
Manufactured by Cytiva
Sourced in Canada, United States
Superdex 75 Increase 10/300 is a prepacked column for size exclusion chromatography. It is designed for the separation and purification of proteins, peptides, and other molecules with a molecular weight range of 3,000 to 70,000 Daltons.
Automatically generated - may contain errors
Lab products found in correlation
Amicon ultra 4 centrifugal filter unit, by Merck Group (1 mentions)
Cyclone plus storage phosphor system, by PerkinElmer (1 mentions)
1260 infinity 2, by Agilent Technologies (1 mentions)
Polygram sil g uv254, by Macherey-Nagel (1 mentions)
Ni nta resin, by Qiagen (1 mentions)
Superdex peptide 10 300, by Cytiva (1 mentions)
Sec 70 gel filtration column, by Bio-Rad (1 mentions)
Pet28a expression vector, by Twist Bioscience (1 mentions)
Kta go protein purification system, by Cytiva (1 mentions)
Molecular imager gel doc xr system, by Bio-Rad (1 mentions)
8 protocols using superdex 75 increase 10 300
1
Preparation of Zn(II)-loaded Metallothionein
2
Radiolabeling of Proteins with Copper-64
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64Ni (98 % enrichment) was electroplated on a Pt/Ir plate (90/10) and irradiated with 12.5 MeV protons on the Tübingen PETtrace cyclotron (GE Healthcare) to produce 64Cu via the 64Ni(p,n)64Cu route. The target was dissolved using concentrated HCl and 64Cu2+ was purified using ion chromatography as described before.58 (link) The obtained radioisotope solution in 0.1 M HCl was buffered with 1.5 volumes of 0.5 M ammonium acetate pH 4.1 before addition of the protein (2 μg per MBq). After 30 min of incubation at 35 °C incorporation of the radioactivity was analyzed by thin layer chromatography (stationary phase: Polygram SIL G UV254, Macherey-Nagel; mobile phase: 0.1 M sodium citrate pH 5) with autoradiographic detection using a phosphor imager (Cyclone Plus Storage Phosphor System, Perkin Elmer). Size exclusion chromatography with radioactivity detector (1260 Infinity II, Agilent; Superdex 75 Increase 10/300, Cytiva) was used to analyze the elution profile of the protein and bound radioactivity. For stripping of the bound radioactivity DTPA (final concentration 0.14 mg/ml) was added to the labeled protein and re-analyzed after incubation at room temperature.
3
Expression and Purification of SARS-CoV-2 Mpro
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The Mpro was expressed and purified
as described previously (Genbank:MN908947.3 ).30 (link) Briefly, the protein was cloned into the pET-SUMO expression vector
resulting in a His-SUMO tag at the N-terminus, transformed into BL21
(DE3) Escherichia coli, induced with 0.5 mM isopropyl
β-d -1-thiogalactopyranoside, and expressed at 32 °C
for 5 h. The protein was purified using Ni-NTA resin (Qiagen, Canada),
followed by SUMO-tag removal using His-tagged SUMO protease (McLab,
United States) and size exclusion chromatography of the final protein
sample on Superdex75 Increase10/300 (Cytiva, Canada). Fractions containing
pure Mpro were pooled, concentrated using an Amicon Ultra-15
(MWCO of 10 kDa), aliquoted, and flash frozen.
as described previously (Genbank:
resulting in a His-SUMO tag at the N-terminus, transformed into BL21
(DE3) Escherichia coli, induced with 0.5 mM isopropyl
β-
for 5 h. The protein was purified using Ni-NTA resin (Qiagen, Canada),
followed by SUMO-tag removal using His-tagged SUMO protease (McLab,
United States) and size exclusion chromatography of the final protein
sample on Superdex75 Increase10/300 (Cytiva, Canada). Fractions containing
pure Mpro were pooled, concentrated using an Amicon Ultra-15
(MWCO of 10 kDa), aliquoted, and flash frozen.
4
Quantitative Analysis of SUMO-SIM Binding
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SUMO and SIM were buffer-exchanged
using a Superdex75Increase 10/300 or Superdex Peptide 10/300 gel filtration
column (Cytiva Life Sciences) into 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA (pH 7.0 or 8.0) or 20 mM MES, 150 mM
KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 6.5). ITC
experiments were performed using a Microcal ITC200 isothermal calorimeter
using 50–100 μM SIM peptide in the cell and 750–1.2
mM SUMO in the syringe. Baseline corrections and integrated heats
were performed using NITPIC.68 (link) Affinity
was determined using a 1:1 binding model in SEDPHAT.69 (link) Reported error is the standard deviation from the Monte
Carlo simulation based on the experimental noise.
using a Superdex75Increase 10/300 or Superdex Peptide 10/300 gel filtration
column (Cytiva Life Sciences) into 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA (pH 7.0 or 8.0) or 20 mM MES, 150 mM
KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 6.5). ITC
experiments were performed using a Microcal ITC200 isothermal calorimeter
using 50–100 μM SIM peptide in the cell and 750–1.2
mM SUMO in the syringe. Baseline corrections and integrated heats
were performed using NITPIC.68 (link) Affinity
was determined using a 1:1 binding model in SEDPHAT.69 (link) Reported error is the standard deviation from the Monte
Carlo simulation based on the experimental noise.
5
Investigating PSL1-SIX8 Protein Interaction
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To investigate the PSL1 and SIX8 interaction in vitro, ~140 µg of PSL118-111 and SIX850-141 individually and ~140 µg PSL118-111 and 140 µg of SIX850-141 together were injected onto a Superdex 75 Increase 10/300 (Cytiva) column pre-equilibrated in 20 mM HEPES pH 7.5, 150 mM NaCl, after a 30 min room temperature incubation. To investigate the residues responsible for the interaction, SIX8_C58S50-141 and PSL1_C37S18-111 mutants were used instead. Samples across the peaks were then analysed by Coomassie-stained SDS-PAGE. To investigate the mode of interaction, PSL1 and SIX8 proteins and mutants at 10 µM were incubated individually or together for 1 hr at room temperature. An unrelated protein with a free cysteine (AvrSr50RKQQC) (Ortiz et al., 2022 (link)) was used to assess the specificity of the PSL1-SIX8 interaction. Proteins were analysed by intact mass spectrometry with or without the addition of DTT as described above.
6
Purification and Characterization of Asparaginyl Endopeptidase
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Size exclusion chromatography (SEC)/fast
protein liquid chromatography was applied as a preparative method
in order to collect larger amounts of the synthetic product. 100 μM
asparaginyl endopeptidase was incubated with 0.5 mM N-terminal and
0.25 mM C-terminal peptidyl substrates in 200 μL of assay buffer
(50 mM sodium acetate pH 5.6, 50 mM NaCl, 1 mM EDTA, and 0.5 mM TCEP)
at 37 °C for 1 h. The reactions were quenched and subjected to
SEC on a Superdex 75 Increase 10/300 (Cytiva) in 10 mM HCl. Absorbance
was recorded at 220 and 280 nm. The product peak was collected and
incubated with 100 mM DTT for 2 h at room temperature. It was then
acidified using 7% HCl (v/v) to a final pH 2.2 and then concentrated
using 3 kDa Amicon Ultra-4 centrifugal filter units. The apo-protein
was again purified on a SEC-70 gel filtration column (Bio-Rad) equilibrated
with 10 mM HCl. To confirm the composition and identity of the product,
the product peak was collected and analyzed by ESI-MS (Figures 5 and S4 ).
protein liquid chromatography was applied as a preparative method
in order to collect larger amounts of the synthetic product. 100 μM
asparaginyl endopeptidase was incubated with 0.5 mM N-terminal and
0.25 mM C-terminal peptidyl substrates in 200 μL of assay buffer
(50 mM sodium acetate pH 5.6, 50 mM NaCl, 1 mM EDTA, and 0.5 mM TCEP)
at 37 °C for 1 h. The reactions were quenched and subjected to
SEC on a Superdex 75 Increase 10/300 (Cytiva) in 10 mM HCl. Absorbance
was recorded at 220 and 280 nm. The product peak was collected and
incubated with 100 mM DTT for 2 h at room temperature. It was then
acidified using 7% HCl (v/v) to a final pH 2.2 and then concentrated
using 3 kDa Amicon Ultra-4 centrifugal filter units. The apo-protein
was again purified on a SEC-70 gel filtration column (Bio-Rad) equilibrated
with 10 mM HCl. To confirm the composition and identity of the product,
the product peak was collected and analyzed by ESI-MS (
7
Recombinant B22R Protein Expression
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The designed B22R gene fragment was codon-optimized, and the synthetic gene was cloned into the pET28a+ expression vector (Twist Biosciences, South San Francisco, CA, USA) and expressed as described below. Induction was performed in BL21 (DE3) cells with 0.1 mM of IPTG and 0.3% L-arabinose. After cellular lysis, the supernatants were harvested by centrifugation at 6000× g for 20 min and purified using fast-flow crude his-trap columns (Cytiva, MA, USA) according to the manufacturer’s instructions. Additional purification was performed by size exclusion chromatography, Superdex Increase 75 10/300 (Cytiva, MA, USA). All the purification steps were performed with an ÄKTA-Go protein purification system (Cytiva, MA, USA). The expression of the protein was confirmed by SDS-PAGE and Western blot using an HRP-conjugated anti-his-tag antibody (dilution 1 in 1000; Merck KGaA, Darmstadt, Germany) and undiluted hyperimmune sheeppox positive serum [35 (link)]. Reactive bands were visualized using the ECL Prime Western-blot-Detection reagent (Cytiva, MA, USA) and detected by the Molecular Imager Gel Doc XR System (BioRad, CA, USA).
8
Purification of Fungal Effector Proteins
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Recombinant SnTox3 73À230 (and mutants), SnToxA 17À178 , FolSIX1 22À284 , FolSIX4 18À242 and FolSIX6 17À225 were cleaved, to remove their putative pro-domains, using recombinant Saccharomyces cerevisiae Kex2 protease (ab96554; Abcam) at a 1 : 200 ratio at room temperature for 48 h. The cleaved SnTox3 proteins were purified further using a Superdex Increase 75 10/300 (Cytiva), pre-equilibrated with 20 mM HEPES pH 7.0 or 7.5 and 150 mM NaCl.
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