Western blot using JUB polyclonal antibody (Cat # PAB9930) shows detection of a 57 KDa band consistent with the expected MW for JUB (arrowhead). Lanes correspond to 1) HeLa nuclear extract, and 2) HeLa, 3) A-431, 4) Jurkat and 5) 293 whole cell lysates. Immunoprecipitation of JUB followed by western blotting may result in cleaner background staining. Approximately 5 ug ofeach preparation was run on a SDS-PAGE and transferred onto nitrocellulose followed by reaction with a 1 : 500 dilution of JUB polyclonal antibody. Detection occurred using a 1 : 5,000 dilution of HRP-labeled Donkey anti-Rabbit IgG for 1 hour at room temperature. Achemiluminescence system was used for signal detection using a 60-sec exposure time. Personal Communication. E. Pugacheva, Fox Chase Cancer Center, Philadelphia, PA.
Western Blot (Transfected lysate)
Western blot using JUB polyclonal antibody (Cat # PAB9930) shows detection of JUB-RFP fusion protein in cell lysates (arrow-head). Lanes correspond to 1) vector only transfection, 2) human JUB-RFP, 3) mouse JUB-RFP, and 4) mock transfection. Approximately 50 ug of each lysate was loaded per lane for SDS-PAGE followed by transfer onto nitrocellulose and reaction with a 1 : 1,700 dilution of JUB polyclonal antibody. Detection occurred using a 1 : 10,000 dilution of IRDye™800 conjugated Gt-a-Rabbit IgG [H&L] for 45 min at room temperature (800 nmchannel, green). Molecular weight estimation wasmade by comparison to prestained MW markers (indicated at left, 700 nm channel, red). IRDye™800 fluorescence image was captured using the Odyssey® Infrared Imaging System developed byLI-COR. IRDye is a trademark of LI-COR, Inc.