Anti flag m2 antibody ab

Cells were lysed in high salt lysis buffer (20 mM Tris-HCl pH 8.0, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, complete protease inhibitor cocktail without EDTA (Roche)) for 30 min on ice. After centrifugation (17,400 × g) at 4°C for 10 min, the supernatant was collected as whole cell lysate. Nuclear extracts were prepared with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Immunoblot analysis was performed with anti-FLAG M2 antibody (Ab) (Sigma-Aldrich), anti-Pax5 Ab (sc-1974, Santa Cruz Biotechnology), anti-C/EBPβ Ab (sc-150, Santa Cruz Biotechnology), anti-β-tubulin Ab (MMS-410P, Covance), anti-MYH9 Ab (sc-47199, Santa Cruz Biotechnology), and anti-chicken Thy28 (cThy28) Ab (kindly gifted by Dr. Compton) [26 (link)].

Yeast cells were suspended in 100 µl (ca. 2.5 volumes of yeast pellet) of a crushing buffer (50 mM sodium phosphate, pH 7.4, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride) and 40 µl of 0.5 mm glass beads (BioSpec Products, Bartlesville, OK, USA, Cat# 11079105), and were crushed with a Bead-Beater (BioSpec Products). Supernatants were recovered after centrifugation at 12,000 rpm for 15 min. Subsequently, 1.2 µg or 6.25 µg of the extract was subjected to Simple Western™ Assay using the “Wes” fully automated system (ProteinSimple, Bio-Techne, MN, USA) with an anti-FLAG M2 antibody (Ab) (Sigma-Aldrich, St Louis, MO, USA, Cat# F1804, RRID: AB_262044) or sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by Coomassie Brilliant Blue (CBB) staining, respectively. Alternatively, the extract (20 µg) was subjected to a general immunoblot analysis with an anti-FLAG M2 Ab as described previously [1 (link)] or SDS–PAGE followed by CBB staining.

Cytoplasmic extracts and nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific). Ten micrograms of cytoplasmic extract from HT1080-derived cells and 8 μg of nuclear extracts from NIH 3T3-derived cells were subjected to immunoblot analysis with anti-FLAG M2 antibody (Ab) (F1804, Sigma–Aldrich, Saint Louis, MO, USA) as described previously [1 (link)].