Anti-Nestin Antibody - Western Blot. Western blot using Affinity Purified anti-Nestin antibody shows detection of a band ~220 kD corresponding to mouse Nestin (arrowhead). Approximately 30 g of MEF whole cell lysate was separated by SDS-PAGE using a 4-20% gradient gel. After transfer onto nitrocellulose, the membrane was blocked and then probed with the primary antibody diluted to 1:2000 overnight at 4°C. The membrane was then washed and reacted with a 1:10000 dilution of IRDye800 conjugated Gt-a-Rabbit IgG [H&L] MX ( for 45 min at room temperature. IRDye800 fluorescence image was captured using the Ddyssey Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.