Melittin, ubiquitin, β-lactoglobulin, and solvents and chemicals not otherwise specified were purchased from Sigma-Aldrich (St. Louis, MO) and used without additional purification. Buffer exchange was performed using BioRad (Hercules, CA) P-6 micro bio-spin columns. Acetylation of primary amine functionalities of Melittin was achieved by incubation of a protein in 2500-fold excess of acetic anhydride in 150 mM ammonium bicarbonate buffer at 298 K. The reaction was quenched after thirty minutes by buffer exchange into ammonium acetate spray buffer. Reduction of disulfide bonds of lactoglobulin was carried out via incubation with 5 mM dithiothreitol at 55°C for 2 hours in 50 mM ammonium acetate.
P6 micro bio spin columns
Manufactured by Bio-Rad
The P6 Micro Bio-Spin columns are a type of size exclusion chromatography columns designed for the rapid purification and desalting of small-volume protein and nucleic acid samples. The columns use a polyacrylamide-based gel matrix to selectively retain sample components based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Ammonium acetate, by Merck Group (2 mentions)
Ammonium hydroxide, by Thermo Fisher Scientific (2 mentions)
Ubiquitin, by Merck Group (1 mentions)
Melittin, by Merck Group (1 mentions)
β lactoglobulin, by Merck Group (1 mentions)
Synapt g2 hdms, by Waters Corporation (1 mentions)
Masslynx software, by Waters Corporation (1 mentions)
4 protocols using p6 micro bio spin columns
1
Protein Modifications and Purification
2
Purification and Nucleic Acid Binding of VP40
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VP40-WT (Δ43, HIS-tagged) was expressed in E. coli and purified as described above, with the SEC peaks yielding ring and monomer/dimer fractions in 10 mM Tris pH 8, 300 mM NaCl. For samples requiring nucleic acid incubation, monomer/dimer was diluted to attain 30 μM VP40,10 mM Tris pH 8,30 mM NaCl and mixed with DNA oligonucleotide (Table S2 ).The molar ratio of VP40 to oligonucleotide was optimized to 1:1 for “HSP,” “HSP18,” and “S2R,” and to 1:4 for “AGGGG.” Incubation was ~24 hours at 4°C. Samples were buffer exchanged into 20 mM ammonium acetate (Sigma) adjusted to pH 8 with ammonium hydroxide (Fisher) using P6 Micro Bio-Spin columns (Bio-Rad) immediately before nMS analysis.
3
Purification and Nucleic Acid Binding of VP40
4
Native IM-MS Protein Structure Analysis
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Both GLB-3a and GLB-3b were buffer-exchanged to an aqueous, volatile ammonium acetate (100 mM NH4OAc, pH 6.8) solution using P-6 micro Bio-Spin columns (Bio-Rad). Protein concentrations were adjusted to 5-10 µM. The samples were infused via in-house produced goldcoated borosilicate glass capillaries using nano-electrospray ionization (nanoESI) into a Synapt G2 HDMS (Waters, Wilmslow, UK) mass spectrometer where native ion mobility-mass spectrometry (IM-MS) experiments were performed. The instrument was operated in mobility mode and the following crucial parameter settings were applied to retain the native, solution-phase structure of the protein: 1.2 kV capillary voltage, 15 V sampling cone, 1 V extractor cone, 10 V and 2 V collision energy in the trap and transfer collision cell respectively, and 45 V trap DC bias.
Pressures throughout the instrument were maintained at 5.0 mbar (backing) and 0.03 mbar (Ar) in the trap and transfer collision cell. Deconvolution of all spectra was performed manually using MassLynx software (Waters, Wilmslow, UK).
Pressures throughout the instrument were maintained at 5.0 mbar (backing) and 0.03 mbar (Ar) in the trap and transfer collision cell. Deconvolution of all spectra was performed manually using MassLynx software (Waters, Wilmslow, UK).
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