Escherichia coli BL21-AI (Invitrogen) were transformed with pDEST15-OTUB1 WT or N22A, respectively, and protein expression was induced by adding 0.2% L-Arabinose for 3h at 37°C. Bacteria were lysed using a Cell Disruptor (TS Series Bench Top, Constant Systems Ltd.) at 35 kpsi in two cycles. Lysates were cleared by ultracentrifugation at 162,000 xg and 4°C for 1 h (Sorvall WX100 Ultracentrifuge) and subsequently affinity purified with Glutathione Sepharose Fast Flow Columns (GSTrap FF, GE Healthcare) in the duo flow system (Bio-Rad). Successful protein expression and purification was verified by Coomassie staining and western blot against OTUB1.
Duo flow system
Manufactured by Bio-Rad
The Duo Flow system is a versatile liquid chromatography instrument designed for efficient protein purification and analysis. It features a dual-pump configuration that enables precise flow control and gradient formation, allowing for accurate and reproducible separation of biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Bl21 ai escherichia coli, by Thermo Fisher Scientific (1 mentions)
Gstrap ff, by GE Healthcare (1 mentions)
Myoglobin, by Bio-Rad (1 mentions)
γ globulin, by Bio-Rad (1 mentions)
Vitamin b12, by Bio-Rad (1 mentions)
Ovalbumin (ova), by Bio-Rad (1 mentions)
Thyroglobulin, by Bio-Rad (1 mentions)
Chondroitin sulfate, by Merck Group (1 mentions)
Heparan sulfate, by Merck Group (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
4 protocols using duo flow system
1
Recombinant OTUB1 Protein Purification
2
Characterization of Recombinant α-Synuclein
3
CXCL13 Binding and Proteolytic Processing
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CXCL13 and CXCL13[1–72] binding to heparin was characterized by loading respective chemokine samples on a 1 ml HitrapTM heparin column (GE Healthcare). Bound CXCL13 and CXCL13[1–72] were eluted using a linear gradient of 0−1.0 M NaCl in 10 mM potassium phosphate, pH 7.5 over 30 min at a flow rate of 1 ml/min and monitored by absorbance at 280 nm on a DuoFlow system (Bio-Rad). The impact of soluble GAGs on CXCL13 processing by Cath-B was determined by performing CXCL13 cleavage experiments in the presence of hyaluronic acid, heparan sulfate or chondroitin sulfate (Sigma).
4
Molecular Weight Characterization of L. delbrueckii Pox
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Prior to analysis of the L. delbrueckii Pox, protein standards were mixed together and applied to the Superdex 200 column (GE healthcare, Chicago, IL) using a duo-flow system (Biorad, Hercules, CA). The protein standards were thyroglobulin (670 kDa), gamma-globulin (150 kDa), ovalbumin (44 kDa), myoglobulin (17 kDa), and vitamin B12 (1.35 kDa). Protein was eluted from the column using a mobile phase of 20 mM Bis-Tris pH 6150 mM NaCl at 0.5 mL/min flow rate. A standard curve was plotted using the peak elution volume for each standard versus the Log10 of the molecular weight in daltons. Eight hundred forty μg of purified L. delbrueckii Pox was incubated in the presence of 50 mM pyruvate, 300 μM TPP, and 15 μM FAD for 10 min at room temperature prior to application to the column. The absorbance at 280 nm was recorded over the elution. One mL fractions were collected after the void volume of 10 mL until the end of the elution. The peak volume for the Pox fractions was used to calculate the molecular weight according to the standard curve.
L. delbrueckii Pox in each fraction from gel filtration was visualized after non-reducing SDS-PAGE analysis with Coomassie staining. For reducing SDS-PAGE analysis, 5% β-mercaptoethanol (Sigma, St. Louis, MO) was included in the sample buffer.
L. delbrueckii Pox in each fraction from gel filtration was visualized after non-reducing SDS-PAGE analysis with Coomassie staining. For reducing SDS-PAGE analysis, 5% β-mercaptoethanol (Sigma, St. Louis, MO) was included in the sample buffer.
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