Mouse monoclonal anti gapdh 6c5

Cultured cells were washed twice with 1 × PBS and then incubated for 1 min in urea buffer (8 M urea, 100 mM NaH₂PO₄ and 10 mM Tris pH 8), scraped, harvested and briefly sonicated. The proteins were subjected to SDS-polyacrylamide gel electrophoresis. The resolved proteins were blotted overnight onto nitrocellulose membranes, which then were blocked in 1 × PBS containing 5% non-fat milk for at least 1 h. The blots were incubated with the following primary anti-human antibodies: rabbit polyclonal anti-N-Myc (C-19; Santa Cruz Biotechnology, Dallas, TX, USA); mouse monoclonal anti-GAP43 (GAP-7B10; Sigma-Aldrich); rabbit polyclonal anti-CDK5 (Cell Signaling Technology, Danvers, MA, USA); mouse monoclonal anti-cyclin A (C-19; Santa Cruz Biotechnology); rabbit polyclonal anti-p27^kip1 (C-19; Santa Cruz Biotechnology); goat polyclonal anti-ChAT (Millipore, Billerica, MA, USA); mouse monoclonal anti-GAPDH (6C5; Millipore) and mouse monoclonal anti-HSP 72/73 (Ab1-W27; Oncogene Science Inc., Cambridge, MA, USA). The membranes were then incubated for 45 min with the appropriate secondary antibody: donkey anti-rabbit IRdye800 (LI-COR Biosciences, Lincoln, NE, USA); donkey anti-mouse IRdye800 (LI-COR) or donkey anti-goat IRdye800 (LI-COR). The membranes were then analysed with a Licor Odyssey Infrared Image System in the 800 nm channel. Blot scan resolution was 150 d.p.i.