b'Pierce ECL Western Blotting SubstrateThermo Scientific Pierce ECL Western Blotting Substrate is a value-priced, entry-level peroxidase substrate for enhanced chemiluminescence (ECL) that directly replaces costlier products without the need to re-optimize conditions. Pierce ECL Western Blotting Substrate provides reliability and performance equivalent to other standard ECL substrates for detection of HRP enzyme activity (Figures 23 and 24). Because the luminol and peroxide reagent formulations are identical to other commercially available substrate products, one can switch to Pierce ECL substrate without needing to optimize probing conditions or incubation protocols.Features: Economicalabout half the cost of other ECL substrates No optimization requiredswitching to Pierce ECL substrate from other entry-level ECL substrates does not require optimization or protocol changes1,800,000 -actin from HeLa cell lysate-galactosidase from Amersham ECL on nitrocellulose E. coli on PVDF1,600,000Pierce ECL MW Protein per well (4050 kDa) MW Protein per well (100120 kDa)1,400,000 Marker 4 2 1 0.5 0.25 (g) Marker 450 225 113 56 20 (ng)Relative intensity unitsPierce1,200,000 ECL substrate1,000,000 1.5-minute exposure 5-minute exposure800,000 AmershamECL reagent600,000 1.5-minute exposure 5-minute exposure400,000Figure 24. Pierce ECL Western Blotting Substrate for detection 200,000 of proteins on nitrocellulose or PVDF membranes. -actin and -galactosidase protein in HeLa cell and E. coli lysates, respectively, were 0 4 g 2 g 1 g 0.5 g 0.25 g detected by western blotting. The membranes were blocked with 5% Protein per well nonfat milk and probed with primary antibody at 1 g/mL. The membranes were washed, then incubated with 0.2 g/mL of HRP-conjugated goat Figure 23. Signal intensity of Pierce ECL Western Blotting Substrateantimouse IgG and washed again. Working solutions of the Pierce ECL is comparable to Cytiva Amersham ECL Western Blotting DetectionWestern Blotting Substrate and Cytiva Amersham ECL Western Blotting Reagent. HeLa cell lysate was separated by SDS-PAGE and transferredDetection Reagent were prepared according to the manufacturers to nitrocellulose membrane to detect -actin. The signal was detected andinstructions and added to replicate membranes for one minute. The analyzed using Kodak 1D Image Analysis Software. Error bars representmembranes were removed from the substrates, placed in plastic sheet 20% difference in relative intensity units. protectors, and exposed to CL-XPosure Film and developed.47'