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Western Blot Analysis Essay

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Immunoprecipitation and western blot analysis
LV-FLAG-HA-T2A-GFP and LV-hZNF804A-FLAG-HA-T2A-GFP constructs were transfected into HEK293 cells with N-terminal V5-tagged APEX1, EIF3K, FEZ1, LGALS1, PDHB, PDZD4, and RPSA overexpression constructs from DNASU. 72 h after the transfection, cells were lysed with cell lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0) and collected by centrifugation at 14 kg at 4°C for 15 min. 3 mg of lysate protein was incubated with 15 l of anti-FLAG M2 beads at 4 °C for 0.5 to 2.5 h with rolling. Each sample was washed for six times with 600 µl of pre-cold TBS, and 15 µl of 2X protein-loading buffer were added for the western blot. Immunoprecipitation and input were …show more content…

Total proteins were prepared from cells of embryonic cortex at different ages (E10–18) and adult brain (2 months) with 500 µl of cell lysis buffer. Cell lysis buffer contains 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM PMSF. After homogenization on ice for 10 min, the supernatant of each sample was collected and stored in aliquots at -80 °C. For each sample, the protein concentration was determined by Bradford assay (Thermo Fisher Scientific). Cell lysate (20–50 µg) was resolved on 12% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF or nitrocellulose membranes. The protein blots were blocked with 5% milk in TBS (10 mM Tris–HCl, pH 8.0, 150 mM NaCl) overnight at 4 °C and incubated with primary antibodies at a dilution of 1 to 1000–2000 in 5% milk prepared in TBS. The secondary antibody was donkey anti-mouse IgG-700 or anti-rabbit IgG-800 (LI-COR). Quantitative western blot was detected by the Odyseey CLx Infrared Imaging System according to the company’s instructions …show more content…

Briefly, yeast cells were transformed with the bait plasmid pHybLex-ZNF804A-Zeo, and selection was maintained with Zeocin. Yeast cells harboring the bait plasmids were transformed with a pre-made HeLa cell-derived Y2H cDNA library constructed in plasmid pJG4-5 in which preys are fused with B42 transcription activation domain (Invitrogen). For screening, transformants were plated on synthetic medium lacking tryptophan, uracil, lysine, and histidine and containing 300 µg/ml Zeocin. After incubation for 3 days at 30°C, growing colonies were selected as primary positives and further tested using a beta-galactosidase colony filter assay, as instructed by the manufacturer. The positive yeast clones were confirmed by DNA sequencing

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