b'WesternBreeze AP chemiluminescent kits Invitrogen WesternBreeze chemiluminescent kits detect proteins that have been immobilized on membranes1 2 3 4 5 1 2 3 4 5kDa(nitrocellulose or PVDF) following western transfer or bound98directly from solution (dot blots). Detection is accomplished with a ready-to-use CDP-Star chemiluminescent substrate52for alkaline phosphatase. Protein bands can be captured either by X-ray film or with a compatible imaging system (Figures 32 and 33).31Blot A: Detected with the WesternBreeze Blot B: Detected with the The WesternBreeze chemiluminescent kits include blockingChemiluminescent Kit, 10-second exposure. WesternBreeze Chromogenic Kit. Figure 32. Human IgG detected with WesternBreeze kits. Human IgG solutions, primary antibody diluent, ready-to-use secondary(h-IgG) was separated on an Invitrogen NuPAGE 412% Bis-Tris gel (with antibody solution (anti-mouse, anti-rabbit or anti-goat),MES SDS buffer) and transferred to a nitrocellulose membrane. The blot ready-to-use chemiluminescent substrate, wash solutions,was probed with a 1:500 dilution of rabbit antihuman IgG and developed with the WesternBreeze Chemiluminescent Kit (anti-rabbit). Lane 1: incubation trays, pre-cut filter papers, and a polyester3 L Invitrogen MultiMark Multi-Colored Standard (no longer available); sheet for even substrate development on the membrane.lane 2: 10 ng of h-IgG; lane 3: 1 ng of h-IgG; lane 4: 100 pg of h-IgG; lane Each kit contains complete reagents for 20 blots. 5: 10 pg of h-IgG.WesternBreeze kitFeatures: 1 2 3 Good signal-to-noise ratiohigh specificity, clean background Time 0 High sensitivityfemtogram levels detectable30 minutes Long signal durationup to 5 days Fastresults in less than 3 hours 1 hour24 hoursFigure 33. Signals achieved with the WesternBreeze chemiluminescent kits. A 53 kDa protein containing a V5 epitope (Invitrogen Positope Control Protein) was separated on a NuPAGE 412% Bis-Tris gel (with MES SDS buffer) then transferred to a PVDF membrane. The blot was probed with a 1:5,000 dilution of mouse anti-V5 primary antibody. Detection was performed using anti-mouse versions of the WesternBreeze detection system with film exposures taken over time. Exposures were two minutes in duration. Time 0 refers to an exposure taken less than 5 minutes after excess substrate removal. Subsequent time points are number of minutes or hours after Time 0. Lanes 13 contained 20 ng, 2 ng, and 200 pg, respectively, of Positope Control Protein. 52'