How to Choose a Secondary Antibody

Detection method	Secondary conjugate	Substrates	Considerations
Enhanced chemiluminescence (ECL)	Horseradish peroxidase (HRP)	Luminol based ECL detection solution	HRP based systems generally more sensitive and easier to work with
	Alkaline phosphatase (AP)	Acridan and 1,2-dioxetane based ECL detection solution
Chromogenic	Horseradish peroxidase (HRP)	3,3’,5,5’-tetramethylbenzidine (TMB), chloro-1-napthol (4CN) or 3, 3’-diaminobenzidine (DAB)	Generally lower sensitivity than ECL and more difficult to work with. Allows multiplexing.
	Alkaline phosphatase (AP)	nitro blue tetrazolium (NBT) or 5-bromo-4-chloro-indolyl phosphate (BCIP)
Fluorescent	Fluorophores (e.g. Alexa Fluor 680)	NA	Can multiplex and signal persists for longer than ECL

Table 1. Summary of detection methodologies for western blotting

 

4.1.1 Enhanced chemiluminescence

Enhanced chemiluminescence (ECL) utilises horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated secondary antibodies that catalyse a reaction which produces light. This is detected by either a super-cooled CCD sensor or x-ray film which enables visualisation of the blot.

HRP conjugated secondaries are most widely used due to their immediate signal generation and higher sensitivity than AP conjugated secondaries. Another consideration when using AP conjugated secondaries is that they are not compatible with phosphate buffers. HRP is inhibited by sodium azide, a commonly used preservative, therefore should not be used with solutions containing NaN₃.

Due to only a single wavelength of light being emitted it is not possible to multiplex unless protein targets are present at different molecular weights whereby multiple targets can be probed for simultaneously on the same membrane (see figure 2 for example). If multiple targets are at a similar molecular weight then it is necessary to strip and re-probe the blot.

 

Figure 2. Example of ECL detection using antibodies for targets at different molecular weights. A mouse monoclonal anti-NFL antibody (HB6433) and mouse monoclonal anti-GAPDH (HB9177) were used to probe a membrane with proteins transferred from various tissue lysates. Visualisation was accomplished using a goat anti-mouse polyclonal antibody conjugated with HRP. Due to the differing molecular weights of NFL and GAPDH this enabled easy multiplexing. Differing signal strengths of NFL and GAPDH required different exposure times meaning that separate panels are required for each target.

 

4.1.2 Chromogenic detection

Chromogenic detection utilises HRP and AP conjugated antibodies that catalyse a reaction leading to a coloured end product which precipitates onto the membrane. Common substrates for HRP are:

• 3,3’,5,5’-tetramethylbenzidine (TMB): Produces a blue product with high signal to noise
• Chloro-1-napthol (4CN): Produces a purple-blue coloured product. However issues with stability and sensitivity
• 3, 3’-diaminobenzidine (DAB): Produces a brown coloured product which can be enhanced using metal ions. Has issues however with stability.

 

Common substrates for AP are:

• Nitro blue tetrazolium (NBT) / 5-bromo-4-chloro-indolyl phosphate (BCIP): Produces an intense purple product which is resistant to fading.

 

AP reactions can be stopped by incubating with an acidic solution therefore allows multiplexing with different coloured reaction products. However AP conjugated secondaries are generally less sensitive than HRP detection systems therefore are much less frequently used. HRP is inhibited by sodium azide, a commonly used preservative, therefore should not be used with solutions containing NaN₃.

 

4.1.3 Fluorescent detection

Fluorescent detection utilises fluorophore conjugated secondary antibodies to detect protein bands. Due to autofluorescence of PVDF at lower wavelengths, fluorophores used for WB detection tend to be highly red-shifted. A list of commonly used fluorophores in ICC/IHC and WB are detailed in table 1. Refer to imaging system specifications to find a combination of fluorophores with excitation/emission wavelengths compatible with your system.

 

Compared with ECL, fluorescent detection is less sensitive however allows easy multiplexing by using fluorophores with sufficiently different excitation/emission wavelengths. The fluorescence signal is also much more stable over time than an ECL signal allowing easy re-imaging while being less variable than ECL detection as it does not rely on a enzymatic reaction which may be influenced by multiple factors.

 

4.2 Immunohistochemistry / Immunocytochemistry

Immunohistochemistry and immunocytochemistry utilise either fluorescence or chromogenic mediated detection. The choice between methods will depend on sample preparation, experimental goals and equipment availability (see table 2 and figure 3 for an overview).