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Choosing Substrate AP vs HRP

Choosing the right substrate for chemiluminescent Western blotting may be a daunting task, given the multitude of options available. The key factors to consider are the level of sensitivity desired, the reporter enzyme used, cost of reagents, versatility, and the nature of the experiment to be performed. One useful starting point in selecting the right chemiluminescent substrate is deciding which reporter enzyme will be used.

In chemiluminescent Western blotting, the reporter enzyme is conjugated to the secondary antibody. The most common reporter enzymes are horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemiluminescent substrates for both HRP and AP are very sensitive and can allow picogram to femtogram level detection.  The maximum signal generated from HRP substrates is very fast, often within 5 minutes. The signal from AP substrates, however, gradually increases over time with a signal plateau around 60 minutes. The signal from AP substrates is very stable and can last up to several days, which can be useful if multiple exposures are needed. Generally, dilution factors for the AP 2° antibodies can be greater than with HRP antibodies, thus saving reagents. The disadvantages of either HRP or AP system are interferences, such as azides for HRP or phosphate buffers for AP. There is also the possibility that endogenous phosphatases will react with AP substrates. Table 1 provides a summary of the differences between choosing an HRP or AP system for chemiluminescent Western blotting.

Michigan Diagnostics, LLC offers several high quality selections for both HRP and AP chemiluminescent substrates. All substrates are highly sensitive to at least low picogram levels and compatible with both nitrocellulose and PVDF membranes. The substrates are also suitable for detection with either film or a CCD camera. The Femto Western HRP substrates come in either the standard two component system or a new single component system for added convenience. The available AP substrates are dioxetane based substrates either with or without fluorescein based additives, for chemifluorescent or standard chemiluminescent detection.

Table 1. Comparison of HRP and AP systems for chemiluminescent Western blotting.

Enzyme Advantages Disadvantages Substrates
  • Very sensitive
  • Economical
  • Immediate max signal generation
  • Inhibited by azides
Femto Western Plus 2 component (Cat. No. FWPD02)

Femto Western Plus Single Component (Cat. No. FWPs02)

  • Very sensitive
  • Long lasting signal (24-48h)
  • Blots can be reactivated
  • Use less 2° antibody
  • Chemifluorescent option available
  • Inhibited by phosphate buffers
  • May react with endogenous phosphatases
Attoglow™ Western

(Cat. No. ATTOW01)

Attoglow™ WesternPLUS

(Cat. No. ATTOWPLUS02)