Sample preparation was carried out in a Coy anaerobic chamber under 90% Ar/10% H2. DT was removed from the solution by buffer exchanging MoFeP into 50 mM TRIS, pH 8.0, 500 mM NaCl using 10-DG desalting columns (BioRad). MoFeP was concentrated to 50 μM using 10-kDa cutoff Microcon filters (EMD/Millipore). Concentrated protein samples were either reduced with 10 mM DT or oxidized with 5 mM IDS. Mutants containing labile iron-centers also had a re-reduced sample prepared in which the IDS-oxidized protein was desalted over another 10-DG column, concentrated, then re-reduced with 10 mM DT. All data were collected on an X-band Bruker EMX spectrometer with a liquid helium cryostat at 5 – 10 K. Spectra were recorded with a modulation frequency of 100.0 kHz and modulation amplitude of 9.8 G. Perpendicular and parallel mode spectra were collected at microwave frequencies of ~9.62 GHz and ~9.39 GHz, and microwave power of 6 mW and 127 mW, respectively. All perpendicular-mode spectra shown in figures were background subtracted.
10dg desalting columns
Manufactured by Bio-Rad
The 10DG desalting columns are designed for the rapid and efficient removal of salts, buffer components, and other low molecular weight substances from protein samples. These columns utilize a size-exclusion chromatography principle to separate the desired protein from unwanted contaminants. The 10DG columns are pre-packed and ready for immediate use, providing a convenient solution for sample preparation prior to further analysis or purification.
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4 protocols using 10dg desalting columns
1
Anaerobic Sample Preparation and EPR Analysis
2
Purification of His-tagged Recombinant Proteins
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Cell pellets were resuspended in lysis buffer (pH 7.5, 50 mM Tris-HCl, 10 mM imidazole, 300 mM NaCl, 1.0 mg/mL lysozyme, 10 µg/mL DNase) and subjected to sonication. The resulting lysate was clarified by centrifugation (13,500 × g for 15 min) and the soluble fraction applied to Ni-Nta agarose (Anatrace). Unbound proteins were removed with lysis buffer, followed by elution of bound proteins with elution buffer (pH 7.5, 50 mM Tris-HCl, 300 mM imidazole, 300 mM NaCl). Proteins were the applied to 10DG desalting columns (Bio-Rad) and eluted in reaction buffer (100 mM sodium phosphate buffer (NaPi), pH 7.5, 150 mM NaCl). Protein purity was confirmed by SDS-PAGE, with concentrations determined by 280 nm absorbance readings, assuming extinction coefficients detailed in Supplementary Data 2 . Protein yields for enzyme purification from a 100 mL E. coli culture are detailed in Supplementary Data 1 .
3
SARS-CoV-2 Protein Purification Protocol
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For S and RBD, four HisTrap Excel columns (column volume = 1 mL or 5 mL) were equilibrated in 10 column volumes (CV) of 20 mM sodium phosphate, 500 mM NaCl pH 7.4. Following parallelized loading (4 × 50 CV for CV = 1 mL; 4 × 200 CV for CV = 5 mL), each column was washed with 60 CV of (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4) and eluted with (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.4) into eight 1 CV fractions. For antibodies, four 1 mL HisTrap Protein A HP (Cytiva) were equilibrated in 10 CV of 20 mM sodium phosphate pH 7.0, loaded in parallel (4 × 10 CV), washed in 30 CV of 20 mM sodium phosphate pH 7.0, and each column was eluted with 0.1 M citric acid pH 3.0 into five 0.85 CV fractions, each fraction containing 0.15 CV of 1 M Tris pH 9.0. All steps were performed at a flow rate calibrated to 1 CV/min at the beginning of the run. Purified protein fractions (8 CV for HisTrap Excel, 5 CV for HisTran Protein A HP) were then buffer exchanged into PBS using either Amicon Ultra-15 centrifugal filter units (Millipore Sigma, 100 kDa cutoff for S, 30 kDa for antibodies, 10 kDa for RBD) or 10DG desalting columns (BioRad). S and RBD proteins, optionally supplemented with 10% glycerol, were filtered and stored at -80°C.
4
Expression and Purification of MaPylRS IFGFF
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For expression of MaPylRSIFGFF, chemically competent E. coli BL21(DE3) were transformed with pET29_MaPylRSIFGFF encoding the protein with a C‐terminal 6‐His tag. A single colony of freshly transformed cells was cultured for 18 h in 10 mL LB medium containing 50 μg/mL kanamycin. Starter culture (5 mL) was used to inoculate 500 mL 2 × YT medium supplemented with 50 μg/mL kanamycin. Cultures were grown at 37°C, 200 rpm to an OD600 ~0.5. Protein expression was induced with the addition of IPTG to a final concentration of 0.1 mM and the culture grown for a further 20 h at 25°C.
The cells were harvested by centrifugation (12 min, 4000 × g), resuspended in lysis buffer (50 mM NaH2PO4, 300 mM pH = 8) and lysed by sonication (1 s on/off, 10 min, 50% amplitude). Cell lysates were cleared by centrifugation (20 min, 27,216 × g) and the clarified lysates subjected to affinity chromatography using Ni‐NTA Agarose (Qiagen). His‐tagged variants were eluted using 50 mM HEPES, 300 mM NaCl, pH 7.5 containing 250 mM imidazole. Purified proteins were desalted using 10DG desalting columns (Bio‐rad) with PBS pH 7.4 and analyzed by SDS‐PAGE and MS.
The cells were harvested by centrifugation (12 min, 4000 × g), resuspended in lysis buffer (50 mM NaH2PO4, 300 mM pH = 8) and lysed by sonication (1 s on/off, 10 min, 50% amplitude). Cell lysates were cleared by centrifugation (20 min, 27,216 × g) and the clarified lysates subjected to affinity chromatography using Ni‐NTA Agarose (Qiagen). His‐tagged variants were eluted using 50 mM HEPES, 300 mM NaCl, pH 7.5 containing 250 mM imidazole. Purified proteins were desalted using 10DG desalting columns (Bio‐rad) with PBS pH 7.4 and analyzed by SDS‐PAGE and MS.
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