BL21(DE3) cells (Invitrogen) transformed with pGEX-KIX plasmid were induced at 37 °C with isopropyl β-d -1-thiogalactopyranoside for 3 h. Cells were lysed in 50 mM Tris–HCl pH 7.3, 150 mM NaCl, 0.1% Tween-20, 1 mM DTT, 5 mM EDTA, supplemented with protease inhibitors described above and sonicated for ten minutes (15 s on, 15 s off, 40% amplitude) using the Misonix probe sonicator (Qsonica, Newtown, CT). Lysate was cleared by centrifugation for 1 h at 21,800 × g at 4 °C. Cleared lysate was incubated with 4 ml glutathione agarose resin slurry (GoldBio) for 1 h at 4 °C to capture GST-KIX. Resin was washed four times with 50 mM Tris–HCl pH 7.4, 150 mM NaCl. KIX domain was cleaved from GST by incubation of resin-bound GST-KIX with 160 U thrombin (GE Healthcare) overnight at room temperature. Resin was centrifuged at 500 × g for 5 min. Supernatant containing cleaved KIX was collected and dialyzed at 4 °C against 50 mM MOPS pH 6.5, 50 mM NaCl, 10% glycerol, 1 μM tris-2-carboxyethylphosphine. Cleaved KIX was purified using a linear gradient of 50 mM to 1 M NaCl by cation exchange chromatography using MonoS 5/50 GL column (GE Healthcare). Fractions containing purified KIX were dialyzed against 50 mM potassium phosphate pH 5.5, 150 mM NaCl, 10 μM tris-2-carboxyethylphosphine, 30% glycerol, and stored at −80 °C.
Mono s 5 50 gl column
Manufactured by GE Healthcare
Sourced in Sweden
The Mono S 5/50 GL column is a cation exchange chromatography column designed for protein purification. It features a bed volume of 5 ml and a column length of 50 mm. The column is composed of a rigid polymethacrylate matrix with sulfopropyl functional groups for the capture and separation of positively charged proteins.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Thrombin, by GE Healthcare (1 mentions)
Bl21 de3 cells, by Thermo Fisher Scientific (1 mentions)
Human ubiquitin, by R&D Systems (1 mentions)
Glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Takara Bio (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Mccoy s 5a medium, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Gentamycin, by Merck Group (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
16 protocols using mono s 5 50 gl column
1
Purification of the KIX Domain
2
Determination of FimCH Impurities by CEX-HPLC
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Example 5
Impurities of FimCH Drug Substance by CEX-H PLC
CEX-HPLC is used for determination of the FimCH complex, unbound FimC and impurities in the final FimCH drug substance. The protein is eluted from a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 M NaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MES buffer, pH 6.2). At T=0 the mobile phase is 100% Buffer A, and at T=22 minutes, the mobile phase is 100% Buffer B. The relative content of the unbound FimC and impurities is determined based on the peak areas. A representative chromatogram is provided in
3
Cation-exchange Chromatography for C3 Variants
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Cation-exchange chromatography was used to identify different C3 populations within native C3, C3 (methylamine), C3 (methylamine F/T), and C3(KSCN). The chromatography was performed using a Mono S 5/50 GL column (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden) coupled to the NGCTM Chromatography System. The flow-rate was set to 0.5 mL/min at RT and a gradient was established from 0 to 0.85 M NaCl in 20 mM Phosphate buffer pH 6.8. Fractions were collected for further analysis.
4
Quantifying FimCH Drug Impurities by CEX-HPLC
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Example 5
Impurities of FimCH Drug Substance by CEX-HPLC
CEX-HPLC is used for determination of the FimCH complex, unbound FimC and impurities in the final FimCH drug substance. The protein is eluted from a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 M NaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MES buffer, pH 6.2). At T=0 the mobile phase is 100% Buffer A, and at T=22 minutes, the mobile phase is 100% Buffer B. The relative content of the unbound FimC and impurities is determined based on the peak areas. A representative chromatogram is provided in
5
Quantification of FimCH Impurities by CEX-HPLC
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Example 5
Impurities of FimCH Drug Substance by CEX-HPLC
CEX-HPLC is used for determination of the FimCH complex, unbound FimC and impurities in the final FimCH drug substance. The protein is eluted from a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 M NaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MES buffer, pH 6.2). At T=0 the mobile phase is 100% Buffer A, and at T=22 minutes, the mobile phase is 100% Buffer B. The relative content of the unbound FimC and impurities is determined based on the peak areas. A representative chromatogram is provided in
6
Synthesis of K48-linked Polyubiquitin Chains
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To generate K48-linked polyubiquitin chains for the experiment shown in Fig. 4c , human ubiquitin (R&D Systems) at a final concentration of 1.7 mM was incubated overnight at 37 °C with 0.333 μM UBE1 (R&D Systems) and 3.33 μM Ube2R1 (R&D Systems) in reaction buffer containing 50 mM Trish-HCl [pH 8.0], 10 mM ATP, 10 mM MgCl2, 1 mM DTT. Ubiquitylation reaction was then diluted ~28-fold with Buffer A (50 mM NH4Ac pH 4.5), and loaded on a MonoS 5/50GL column (GE Healthcare). Ubiquitin chains were eluted with a linear gradient (5–100%) of Buffer B (50 mM NH4Ac pH4.5, 1 M NaCl), and peak fractions were combined, concentrated on Amicon Ultra-15 3 K concentrator (Millipore) and simultaneously exchanged into storage buffer (10 mM Tris-HCl pH 7.6), then flash frozen in liquid nitrogen and stored at −80 °C.
7
Affinity Purification and Biochemical Analysis
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Heat-inactivated fetal bovine serum (FBS), geneticin, Precast 4–20% Tris-glycine gels, L-glutamine, antibiotic/antimycotic and penicillin/streptomycin were from Thermo Fisher Scientific (Waltham, MA). Talon metal affinity resin and puromycin were from Clontech Laboratories (Takara Bio USA, Inc.). Gentamycin, N-Ethylmaleimide (prepared as a 0.4 M stock solution in ethanol), Anti-Flag M2 affinity gel, cycloheximide (prepared as a 100 mM stock solution in DMSO), MG132 (prepared as a 100 mM stock solution in DMSO) and hydrogen peroxide solution (diluted in H2O) were from MilliporeSigma. McCoy’s 5A medium, Dulbecco’s modified Eagle’s medium (DMEM), α-MEM and minimal essential medium (MEM), as well as the Glutathione agarose, Pierce IP lysis buffer and RIPA buffer, were from Thermo Fisher Scientific. Dimethyl sulfoxide (DMSO) was from Fisher Biotech (Fair lawn, NJ). Fugene 6 transfection reagent and protease inhibitor cocktail tablets were from Roche (Indianapolis, IN). NADH was from Alfa Aesar Chemicals of Thermo Scientific (Tewksbury, MA). Mono-S 5/50 GL column, Superdex 200 increase 10 × 300 GL column and glutathione sepharose 4B were from GE Healthcare (Piscataway, NJ). All of the primers were synthesized and purified by Thermo Fisher Scientific (Waltham, MA).
8
Determination of FimCH Impurities by CEX-HPLC
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Example 5
Impurities of FimCH Drug Substance by CEX-H PLC
CEX-HPLC is used for determination of the FimCH complex, unbound FimC and impurities in the final FimCH drug substance. The protein is eluted from a GE Healthcare Mono S 5/50 GL column using a gradient of 0.3 M NaCl in 20 mM MES buffer, pH 6.2 (Buffer B) (Buffer A is 20 mM MES buffer, pH 6.2). At T=0 the mobile phase is 100% Buffer A, and at T=22 minutes, the mobile phase is 100% Buffer B. The relative content of the unbound FimC and impurities is determined based on the peak areas. A representative chromatogram is provided in
9
Purification and Characterization of Vaa Venom
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Crude Vaa venom was provided by the Institute of Immunology, Zagreb, Croatia. Lyophilized venom was kept at −20 °C. Before the analysis, it was dissolved in 50 mM Tris, 2 mM CaCl2, 300 mM NaCl, pH 7.0 (buffer A). Fifteen milliliters of the crude venom solution (i.e., 1 g of the venom) was applied to a Sephacryl S-200 Superfine column (100 cm × 4 cm) (GE Healthcare BioSciences AB, Uppsala, Sweden). Gel chromatography was performed at a constant flow rate of 33.6 mL/h. The concentration of proteins in the mobile phase was monitored by measuring their absorbance at 280 nm (A280). The B1 fraction was dialyzed against 20 mM MES, 2 mM CaCl2, pH 6.5 (buffer B), and further fractionated on a Mono S 5/50 GL column of the FPLC system (ÄKTA, GE Healthcare Life-Science, Uppsala, Sweden), equilibrated with the same buffer. The column-retained material was eluted using a linear gradient of NaCl (0 to 1 M) in the buffer B. VaaSP-VX was eluted from the column, concentrated, and dialyzed against buffer B, at pH 7.5.
10
Phosphorylation of Rab8a by MST3 Kinase
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Full length GST-MST3 produced in insect cells (DU30889) was obtained from MRC-PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk/reagents-proteins/overview ). Rab8a was incubated with GST-MST3 at molar ratios between 4:1 to 9:1 (substrate:enzyme). Typical concentrations of Rab8a were 1-3 mg/ml, while the concentration of MST3 was 1 mg/ml in a total volume between 2-15 ml. The buffer of the reaction was adjusted to 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgCl2 and 2 mM ATP, pH 7.5. The reaction mixture was incubated at room temperature overnight (12-18 hours). To separate pRab8a from the non-phosphorylated form, the reaction mixture was dialyzed against low-salt ion exchange buffer (10 mM MES, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT, pH 5.2) for two hours and then loaded onto a MonoS 5/50 GL column (GE Healthcare) equilibrated to the low-salt ion-exchange buffer. Elution of pRab8a was performed by running a 50% gradient from low- to high-salt buffer (10 mM MES, 1 M NaCl, 5 mM MgCl2, 1 mM DTT, pH 5.2) over 30 column volumes (Figure S2 ). The phosphorylation of Rab8a1-181 was confirmed by PhosTag gel electrophoresis. In order to stabilize pRab8a, the pH was adjusted to pH 7.5 immediately after elution from the ion-exchange column.
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