Enzymes such as AP and HRP convert several substrates to a colored precipitate. As the precipitate accumulates on the blot, a colored signal develops that is visible on the blot (Figure 4.9A). The enzyme reaction can be monitored and stopped when the desired signal over background is produced. Colorimetric detection is easier to use than any film-based detection method, which must be developed by trial and error, and uses costly materials such as X-ray film and darkroom chemicals.
Colorimetric detection is typically considered a medium-sensitivity method, compared to radioactive or chemiluminescent detection.
Fig. 4.9. Mechanisms of detection chemistries.
In each method of western blot detection, a detectable signal is generated following binding of an antibody specific for the protein of interest. In colorimetric detection (A), the signal is a colored precipitate. In chemiluminescent (B) and bioluminescent (C) detection, the reaction itself emits light. Chemiluminescent and bioluminescent detection are distinguished by the source of the substrate. In chemifluorescent detection (D), the product is fluorescent. Fluorescent detection, autoradiography, and immunogold detection record the signal generated by a labeled secondary antibody. In fluorescent detection, the antibody is labeled with a fluorophore, while in auto- radiography, it is labeled with a radioactive isotope (E). In immunogold detection (F), the secondary antibody is labeled with gold, and signal is generally enhanced by silver precipitation.
E.Fluorescence/ autoradiography F.Immungold detection C.Bioluminescence A.Colorimetric detection B.Chemiluminescence D.Chemifluorescence Target protein Primary antibody Secondary antibody Enzyme conjugate Substrate Product
Label (radiolabel or fluorophore) Emitted light or radiation Precipitate
However, Bio-Rad has colorimetric systems that offer very high sensitivity equal to detection by chemiluminescence (Table 4.8).
Colorimetric HRP Systems
Colorimetric HRP systems were the first enzyme-conjugates used for immunological detection of blotted proteins. The advantage of HRP systems was that both the enzyme conjugate and colorimetric detection substrates were economical. The most common color substrates for HRP are 4-chloro-1-naphthol (4CN) (Hawkes et al. 1982) and 3,3'-diaminobenzidine (DAB) (Tsang et al. 1985) (Figure 4.10). HRP colorimetric detection systems are not as
sensitive as AP colorimetric detection systems. Fading of blots upon exposure to light, inhibition of HRP activity by azide, and nonspecific color precipitation are additional limitations of HRP colorimetric detection systems.
Opti-4CN Substrate and Detection Kits
Colorimetric HRP detection with 4CN presents very low background and a detection sensitivity of about 500 pg of antigen. Bio-Rad’s Opti-4CN kit improves this detection sensitivity to 100 pg. Opti-4CN is available as a premixed substrate kit or combined with an HRP- conjugated antibody in a detection kit.
Sensitivity
Substrates
Comparative cost Stability of stored blots Restrictions HRP 500 pg (4CN and DAB) 1–3 pg (Immun-Star™HRP) 4CN — purple DAB — brown Luminol — emits light Least expensive Poor for 4CN and DAB Good for Immun-Star kits Azide inhibits peroxidase activity
AP
100 pg (Immun-Blot®) 10 pg (Immun-Star AP) 10 pg (Immun-Blot amplified AP) BCIP/NBT — purple
CDP-Star— emits light More expensive Good
Endogenous phosphatase activity also detected
Table 4.7. Comparison of detection reagent systems.
Detection Detection
Method Substrate Sensitivity Signal Color Product Options Advantages Disadvantages
Colorimetric HRP 4CN 500 pg Purple • Dry powder, liquid • Fast color development, • Results fade over time; substrate, Immun-Blot kits low cost, low background azide inhibits
enzyme activity
DAB 500 pg Brown • Dry powder • Fast color development, • More safety
low background precautions than for other substrates • Azide inhibits
enzyme activity Opti-4CN 100 pg Purple • Liquid substrate, • High sensitivity, nonfading • More expensive
Opti-4CN kit color, low background than 4CN
Amplified 5 pg Purple • Amplified Opti-4CN kit • Best sensitivity available; • More steps than
Opti-4CN no extra materials (such unamplified protocol
as X-ray film) needed
Colorimetric AP BCIP/NBT 100 pg Purple • Dry powder, liquid • Stable storage of data • Detects endogenous
substrate, Immun-Blot kits phosphatase activity
Amplified 10 pg Purple • Amplified AP • High sensitivity • More steps than
BCIP/NBT Immun-Blot kit unamplified protocol
Amplified Opti-4CN Substrate and Detection Kits
Amplified Opti-4CN substrate and detection kits are based on proprietary HRP-activated amplification reagents from Bio-Rad. These kits allow colorimetric detection to 5 pg, which is comparable to or even exceeds the sensitivity that is achieved with radiometric or chemiluminescence systems, without the cost or time involved in darkroom
development of blots.
Immun-Blot HRP Assay Kits
Immun-Blot assay kits provide the reagents required to perform standard HRP/4CN colorimetric detection on western blots with the added convenience of premixed buffers and enzyme substrates.
Premixed and Individual HRP Colorimetric Substrates
Premixed enzyme substrate kits and development reagents, including powdered 4CN and DAB color development reagents, are also available. The premixed kits are convenient and reliable and reduce
exposure to hazardous reagents used in the color development of protein blots.
Fig. 4.10. Colorimetric detection options with HRP.
DAB and 4CN are commonly used chromogenic substrates for HRP. In the presence of H2O2, HRP catalyzes the oxidation of the substrate into a product that is visible on a blot. Left, reaction with DAB; right, reaction with 4CN.
H2N NH2 H2N NH2 NH + HRP–O + H2O H2N NH2 HRP–O DAB NH H2N NH2 –1e–
–1e– Quinone iminium cation radical
n Polymerization to complex brown precipitate •• HN•+ HN•+ •• HRP + H2O2 OH CI HRP + H2O2 O CI H Insoluble purple product 4CN H2O
Fig. 4.11. AP colorimetric development. In the colorimetric system, AP catalyzes the substrates BCIP and NBT to produce a colored precipitate visualizing the protein on a western blot. First the dephosphorylation of BCIP by AP occurs, yielding a bromochloro indoxyl intermediate. The indoxyl is then oxidized by NBT to produce an indigoid dye (purple precipitate). The NBT is also reduced by the indoxyl, opening the tetrazole ring to produce an insoluble diformazan (blue precipitate). The combination of the indigoid dye of the BCIP and the insoluble formazan of the NBT forms a purple-blue colored precipitate.
Immun-Blot AP Assay Kits
The Immun-Blot AP assay kits provide the essential reagents to perform colorimetric detection on western blots with the added convenience of premixed buffers and enzyme substrates. All kit components are individually quality-control tested in blotting applications. Included in each kit is an instruction manual with a thoroughly tested protocol and troubleshooting guide that simplifies immunological detection.
Immun-Blot Amplified AP Kit
Increased sensitivity in western blot experiments can be achieved by utilizing an amplified AP procedure (Bayer and Wilchek 1980, Chaiet and Wolf 1964, Guesdon et al. 1979, Hsu et al. 1981). This detection system begins by using a biotinylated secondary antibody. Relying on the specific binding properties of biotin and avidin, a complex of streptavidin and biotinylated AP is then added to the membrane. Because streptavidin will bind more than one molecule of biotin, the initial site of the primary antibody-to-antigen binding is
Colorimetric AP Systems
Colorimetric AP systems use soluble 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitroblue Tetrazolium (NBT) as substrates to produce a stable reaction product that will not fade (Figure 4.11). AP can be easily inactivated by exposure to acidic solutions. Multiple probing of the same membrane with alternative antibody probes is easily performed using substrates that produce different colors, such as blue and red (Blake et al. 1984, Turner 1983).
O CI Br N H O CI Br H N Indigoid dye (purple precipitate) C C O O N N N+ N N+ –O O N+ N N N N+ O O– C C O O N N N H N N+ –O O N H N N N N+ O O– Insoluble diformazan (blue precipitate) NBT O CI Br N H H H + 2 BC indoxyl intermediate AP + H2O – OPO3 2– P O ONa ONa O CI Br N H BCIP
effectively converted into multiple AP binding sites available for color development (Figure 4.12). Color development is
performed using conventional AP substrates, as discussed previously. The Immun-Blot amplified AP kit increases the detection sensitivity of colorimetric western blotting to ≥10 pg of protein.
Premixed and Individual AP Colorimetric Substrates
Premixed enzyme substrate kits are convenient and reliable and reduce exposure to hazardous reagents.