Quantitative Protein Analysis of Spinal Cords

Spinal cords were isolated from control and mutant E18.5 embryos. Tissue lysates were prepared and diluted to protein concentration of 0.4 μg/μl using sample preparation kit (ProteinSimple). Tissue lysates were examined using automated western blot system, WES System (ProteinSimple) as per manufacturer’s protocol. The following primary antibodies were used for analysis: ZPR1 (Clone LG-C61), Flag M2 (#F1804, Sigma), HoxA5, HoxC5, α-tubulin (#T8203, Sigma) and β-Actin (#A5316, Sigma). Data analysis and quantitation of protein levels were performed using Compass Software (Protein Simple). For standard immunoblot method, cell lysates were prepared from control and transfected HeLa cells using Triton lysis buffer^{5 (link)}. Proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane (Millipore). Proteins were detected using primary antibodies (1:100), ZPR1 (Clone LG-C61), Flag M2 and α-tubulin (#T6074, Sigma) followed by HRP-conjugated donkey anti-mouse or rabbit IgG (1:5000) secondary antibodies. Chemiluminescence and quantitation of immunoblots was performed using ImageQuant LAS4000. The relative levels of proteins (mean ± s.e.m.) normalized to either actin or tubulin, are presented as bar graphs.