The Trs120-3xFLAG–tagged S. cerevisiae (44 liters) were grown in YPD (yeast extract peptone dextrose) medium at 30°C overnight until an OD600 (optical density at 600 nm) reached 5 to 6. The cells were pelleted by centrifugation and resuspended using the lysis buffer [20 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 10% glycerol, 1% CHAPS, 1 mM EDTA, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF)] supplemented with a protease inhibitor cocktail (Roche). The cell pellets were frozen by liquid nitrogen and then lysed with Freezer Mill 6875 (SPEX CertiPrep). The lysate was centrifuged at 142,400g for 40 min using a Type 45 Ti rotor (Beckman). All supernatants were incubated with anti-Flag affinity resin (Sigma-Aldrich) at 4°C for 2 hours. The resin was then washed by the washing buffer [20 mM Hepes-NaOH (pH 7.4), 300 mM NaCl, 10% glycerol, 0.1% CHAPS, 1 mM EDTA, 2 mM DTT] and eluted with the washing buffer supplemented with Flag peptide (0.1 mg/ml; Sigma-Aldrich). The eluate was concentrated and applied to Superose 6 Increase 3.2/300 (GE Healthcare) equilibrated with the sample buffer [20 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 2 mM DTT, 0.05% digitonin]. The peak fractions were analyzed by SDS-PAGE and the MS analysis.
Superose 6 increase 3.2 300
Manufactured by GE Healthcare
Superose 6 Increase 3.2/300 is a size exclusion chromatography column designed for the separation and purification of molecules. It is suitable for analytical and preparative applications. The column has a bed volume of 3.2 mL and a bed height of 300 mm.
Automatically generated - may contain errors
Lab products found in correlation
Type 45 ti rotor, by Beckman Coulter (1 mentions)
Anti flag affinity resin, by Merck Group (1 mentions)
Flag peptide, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
6875 freezer mill, by SPEX (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
3 protocols using superose 6 increase 3.2 300
1
Purification of Trs120-3xFLAG Protein
2
Purification of Trs120-containing Complex
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A yeast strain, in which Trs65 gene was deleted from the genome and contained C-terminal 3xFlag-tagged Trs120, was constructed and named Trs65del.
The construction of 6xHIS-GFP-Trs65 driven by GAL1 promotor was transformed into yeast strain Trs65del. Twelve liters of transformed yeast cells was cultured in minimal medium lacking leucine with supplement of raffinose until the OD600 reached logarithmic growth phase, and then galactose was added to the final concentration of 100 mM. After 6 hours of culture, the cells were pelleted and proteins were purified by anti-Flag affinity resins (Sigma-Aldrich). Anti-HIS antibodies (ZSGB-BIO) were added at a 1:1.5 molar ratio (antibody to protein) into the purified protein and incubated for 30 min at room temperature. The sample was concentrated and applied to Superose 6 Increase 3.2/300 (GE Healthcare). The peak fraction was analyzed by negative-stain EM.
The construction of 6xHIS-GFP-Trs65 driven by GAL1 promotor was transformed into yeast strain Trs65del. Twelve liters of transformed yeast cells was cultured in minimal medium lacking leucine with supplement of raffinose until the OD600 reached logarithmic growth phase, and then galactose was added to the final concentration of 100 mM. After 6 hours of culture, the cells were pelleted and proteins were purified by anti-Flag affinity resins (Sigma-Aldrich). Anti-HIS antibodies (ZSGB-BIO) were added at a 1:1.5 molar ratio (antibody to protein) into the purified protein and incubated for 30 min at room temperature. The sample was concentrated and applied to Superose 6 Increase 3.2/300 (GE Healthcare). The peak fraction was analyzed by negative-stain EM.
3
Characterizing Protein Complex Formation
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Size exclusion chromatography studies were carried out sequentially over the course of one day using a Superose 6 Increase 3.2/300 (GE Healthcare) column and a buffer containing 20 mM Hepes pH7.5, 150 mM NaCl, and 2 mM DTT. The samples were prepared by mixing substoichiometric amounts of HBS1L3 with either 100 pmol hSKI (blue chromatogram in Figure 7 A), or 100 pmol hEXO10c (red chromatogram in Figure 7 A), or 100 pmol hSKI and 100 pmol hEXO10c (purple chromatogram in Figure 7 A). In order to show that HBS1L3 is required to bridge the interaction between hSKI and hEXO10c and to from a higher order complex, 100 pmol hSKI with 100 pmol hEXO10c were mixed in absence of HBS1L3 (green chromatogram in Figure 7 A). The samples were adjusted to 35 μl with buffer and incubated at 4°C for 15 min before injecting them onto the column. The absorbance at 280 nm was recorded in the chromatograms. For comparison of the retention volumes of the different complexes, the data was normalised and plotted in R using the tidyverse collection of R packages. Peak fractions of the individual gel filtration runs were TCA precipitated and analysed by SDS-PAGE on a NuPAGE 4-12% Bis-Tris gel (Thermo Fisher Scientific).
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