cell line: HEK293T recombinant protein expression: V5-DDX56
Treatment protocol
HEK293T cells were transfected with 15 µg of the respective expression constructs using Calcium phosphate.
Growth protocol
HEK293T cells were grown in 15 cm2 dishes at 37°C with 5% CO2 in DMEM medium supplemented with 10% fetal calf serum, 2 mM L-Glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.
Extracted molecule
protein
Extraction protocol
Cells were lysed in 50 mM HEPES-KOH, pH 7.7, 1 mM MgCl2, 300 mM NaCl, 0.8 % Triton X-100, 5 mM NaF, 0.1 % Sodium deoxycholate, 2 mM β-mercaptoethanol, 1x protease inhibitor mixture tablet (Roche). Cleared lysates were immunoprecipitated using anti-V5 agarose beads (Roche) at 4 °C for 3 h. Beads were washed in TBS-T supplemented with 1 mM MgCl2, 2 mM β-mercaptoethanol and 1x protease inhibitor mixture tablet (Roche), followed by elution in the same buffer using 200 µg/ml V5 peptide.
The protein microrrays (ProtoArray v5.1, Invitrogen) were incubated for 1 h at 4 °C in Blocking Buffer I (PBS, 0.1 % Tween-20, 1 % BSA). The arrays were washed three times at 4 °C in Blocking Buffer II (TBS supplemented with 0.5 % Triton X-100, 1 mM β-mercaptoethanol, 5 % Glycerol, 2 mM MgCl2, 1 % BSA), placed at room temperature and incubated for 1 h with 600 µl of ~ 0.5 µM of immunopurified V5-DDX39, V5-DDX56 or V5-GFP. The arrays were then washed twice in Blocking Buffer II at 4 °C and incubated with 1:1000 α-V5 tag monoclonal antibody (Thermo Fisher, R960-25) for 2 h at 4 °C. After three additional washes in Blocking Buffer II, the microarrays were incubated with anti-mouse secondary antibody Alexa Fluor 680 (Thermo Fisher) for 1 h at 4 °C and washed again three times. Finally, the arrays were rinsed 3 times in water and dried by spinning at 300 g for 5 min.
Scan protocol
The microarrays were scanned in an Odyssey scanner (LI-COR) using the following parameters: Channel 700, Grid = 8x3, 21.17, Intensity = 5, Offset = 0.
Description
Array ID: 0000088692
Data processing
ProtoArray signal data was determined with GenePix Pro v5.0.0.49 (Molecular Devices) and then processed following the manufacturer's recommendations using ProtoArray Prospector v5.2 (Invitrogen) with default settings.
