b'Chemiluminescent HRP substrates to confirm the presence of target proteins, purpose-built Chemiluminescent substrates for HRP are typicallywestern blot imaging instrumentation offers the advantages two-component systems consisting of a stable peroxideof quantitative analysis, simple exposure time adjustments, solution and an enhanced luminol solution. In most cases,higher sensitivity, greater resolution, and a larger dynamic to make a working solution, equal volumes of the tworange than film. The choice of HRP chemiluminescent components are mixed together. When incubated with asubstrate should be based on abundance of the target blot on which HRP-conjugated antibodies (or other probes)protein of interest, abundance of sample containing the are bound, a chemical reaction emits light at 425 nm thattarget protein, quality of the antibodies, and the level can be captured with X-ray film or an imaging instrumentof sensitivity and type of instrumentation available for that detects chemiluminescence. Although X-ray filmdetection (Tables 18 and 19).provides qualitative and semiquantitative data and is useful Table 18. Properties of Thermo Scientific HRP-based chemiluminescent substrates.Pierce ECL WesternSuperSignal West Pico PLUSSuperSignal West DuraSuperSignal West Atto Blotting Substrate Chemiluminescent Substrate Extended Duration Substrate Ultimate Sensitivity SubstrateDetection limit Low- to mid-picogram Low-picogram to Mid-femtogram Low-femtogram to high-attogramhigh-femtogramRecommended1: 1:1,0001:5,000 1: 1:1,0001:5,000 1: 1:1,0001:50,000 1: 1:1,0001:5,000antibody dilution 2: 1:1,0001:15,000 2: 1:10,0001:100,000 2: 1:50,0001:250,000 2: 1:100,0001:250,000Advantages Low cost, easy to switchSuperior sensitivity and intensityGood sensitivity with longMost sensitive substrate for HRP from other entry-levelwith longer duration than otherduration and linearity fordetection, with high signal-to-ECL substrates entry-level ECL substrates sensitive quantitation noise ratioSelect when Target and sample areRoutine western blots; workingPerforming quantitative westernTarget is very low in abundance abundant with new protein target whenblots or long duration is needed or sample is limitedwestern blotting conditions are not yet optimizedCat. No. 32106 34578 34075 A38554Table 19. Choosing the correct HRP-based substrate for your application needs.ConditionsSample Target protein Antibody Recommended substrate Primary Secondary Special notesSample isTarget protein isAntibody qualityPierce ECL Western Blotting Substrate 1:1,000 1:15,000abundant abundant is goodSample isTarget proteinAntibody quality isSuperSignal West Pico PLUS1:1,000 1:100,000 If no bands are seen, the blot can be washed abundant abundance isunknown Chemiluminescent Substrate and re-imaged with SuperSignal West unknown Atto substrateSample isTarget proteinAntibody qualitySuperSignal West Atto Ultimate1:1,000 1:100,000abundant abundance is low is poor Sensitivity SubstrateSample isTarget proteinAntibody qualitySuperSignal West Atto Ultimate1:2,000 1:250,000abundant abundance is low is good Sensitivity SubstrateSample isTarget proteinAntibody quality isSuperSignal West Atto Ultimate1:5,000 1:100,000 Assumes normal primary usage is 1:1,000abundant abundance is low limited Sensitivity SubstrateSample isTarget protein isAntibody quality isSuperSignal West Atto Ultimate1:20,000 1:250,000 Assumes normal primary usage is 1:1,000abundant abundant limited Sensitivity SubstrateSample isTarget protein isAntibody qualitySuperSignal West Pico PLUS1:1,000 1:100,000 Alternatively, could load less and use limited abundant is good Chemiluminescent Substrate SuperSignal West Atto substrate at the same antibody concentrationsSample isTarget proteinAntibody quality isSuperSignal West Pico PLUS1:1,000 1:100,000 If no bands are seen, the blot can be washed limited abundance isunknown Chemiluminescent Substrate and re-imaged with SuperSignal West unknown Atto substrateSample isTarget proteinAntibody qualitySuperSignal West Atto Ultimate1:1,000 1:250,000limited abundance is low is good Sensitivity SubstrateSample isTarget proteinAntibody qualitySuperSignal West Atto substrate 1:1,000 1:100,000limited abundance is low is poor45'