HckFLAG V284C was integrated into the Flp-In T-REx system (Thermo) as described in the product documentation and maintained with 50 μg/mL hygromycin after selection. Flp-In T-REx HEK293 cells stably expressing HckFLAG V284C were grown in custom -Lys/-Arg DMEM (Caisson Labs) supplemented with 10% dFBS (Sigma), 200 μg/ml proline and SILAC amino acids (0.2 mM natural isotope abundance Lys/Arg for light label, 0.2 mM Lys8/Arg10 for heavy label; Cambridge Isotope Labs). Cells were grown for at least 5 cell doublings in SILAC medium and harvested after reaching 90% confluency and 24 h induction with 0.5 mg/mL dox. For harvesting, cells were rinsed twice with ice cold PBS and lysed in 750 μL of mod. RIPA buffer 2 (50 mM Tris, pH 7.8, 150 mM NaCL, 10 mM NaF, 0.25% Na-deoxycholate, 1% NP-40 and 5% glycerol containing Halt Protease Inhibitor Cocktail (100x, Thermo Scientific), and Phosphatase inhibitor cocktail 2 and 3 (Sigma)). The ice-cold lysate was vortexed five times intermittently for 3 s and clarified by centrifugation at 21,000xg and 4 °C for 20 min. The protein content of the samples was determined using the Pierce 660 nm Assay Reagent (Thermo), then snap frozen in liquid nitrogen and stored at −80 °C until used (Golkowski et al., 2017 (link)). Relates to Figure 4H and S3G .
Pierce 660 nm assay reagent
Manufactured by Thermo Fisher Scientific
The Pierce 660 nm Assay Reagent is a quantitative protein assay used to determine the total protein concentration in a solution. It is a colorimetric assay that utilizes the 660 nm wavelength to measure the protein content of samples.
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Lab products found in correlation
Phosphatase inhibitor cocktail 2 and 3, by Merck Group (1 mentions)
Halt protease inhibitor cocktail 100x, by Thermo Fisher Scientific (1 mentions)
Flp in t rex system, by Thermo Fisher Scientific (1 mentions)
Pd 10 column, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Dnase, by Roche (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Criterion xt bis tris precast gel 4 12, by Bio-Rad (1 mentions)
Pefabloc sc, by Roche (1 mentions)
Coomassie brilliant blue r 250 staining solution, by Bio-Rad (1 mentions)
Q700 sonicator, by Qsonica (1 mentions)
3 protocols using pierce 660 nm assay reagent
1
Stable Expression and Lysis of Hck^FLAG V284C
2
Purification of His6-MBP-GNAT Fusion Proteins
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The His6‐MBP‐GNAT protein constructs were expressed in E. coli BL21(DE3)pLysS as described above. Afterward, cells were harvested, resuspended in buffer (50 mM Tris–HCl, pH 8, 500 mM NaCl, 5 mM MgCl2, protease inhibitor cocktail [Sigma‐Aldrich]), and disrupted using a French Press. The cell extract was then complemented with 5 mM DTT and 50 units of DNAse (Roche), before being loaded on a Protino Ni‐NTA affinity chromatography matrix (Macherey‐Nagel). His6‐MBP‐GNAT2 or His6‐MBP‐GNAT10 was eluted with 500 mM imidazole and desalted by gel filtration using PD‐10 columns (GE Healthcare). For storage, the protein preparations were buffered in 100 mM Tris–HCl (pH 8). Protein concentration was determined with the Pierce™ 660 nm Assay Reagent (Thermo Fisher).
3
Isolation of Insoluble Protein Aggregates
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Insoluble protein aggregates were isolated as described (Rand and Grant, 2006), with minor adjustments. The protocol was scaled up to suit the harvesting of 35 OD600 units of cells. Pefabloc SC (Roche) was used instead of phenylmethylsulfonyl fluoride in the lysis buffer. After freezing and thawing the cells they were immediately disrupted by a minibead beater (Fastprep) at 5.5 m/s for 4x30 sec at 4°C. Cells were kept on ice for 5 min between each beating. Removal of intact cells was accomplished by centrifugation at 1180 x g for 15 min. Protein concentration was determined using Pierce 660 nm Assay Reagent (Thermo Scientific) and adjusted before isolation of protein aggregates as well as verified by running reduced samples on Criterion XT Precast Gel 4–12% Bis-Tris (BioRad) and staining with Coomassie Brilliant Blue R-250 staining solution (BioRad). The subsequent centrifugations were all done at 21130 x g for 20 min. Insoluble fractions were resuspended in detergent washes by 5 x 5 sec sonication at amp 40 with 15 sec rest between intervals using Q700 Sonicator (QSonica, LLC. Newtown, USA). The insoluble fractions were added to reduced protein loading buffer, loaded on Criterion XT Precast Gel 4–12% Bis-Tris (BioRad) and visualized by silver staining with Pierce Silver Stain Kit (Thermo Scientific).
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