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expo$urc of pcrconnel to radiation, high disposal costs, and cnvironn\cntal conccms. Lastly, chcmdumincsccnt methods silów stripping and u rcprobing of hlots multiple timcs, which js not. poss\hlc with colorimctric dctcction tcchntąues. ‘

Biolumlnescent Detectton BioUiminesccncc is thc phenoiucnón of light cmission hy many organisms. Bioluminescent I sysiems differ ln thc structure and function of thc enzymes and cofnctors involvcd in the procesu as [wcll as thc mcchanism of thc lighi-generatmg I rtractions.

Biolumincscencc can be cxploited as a dctcction mcthisl for western blots. Bioluminescent dctcction I involves incuhation of thc membranę with thc bound antigen/antibody-cnzyme complex in a bioluminogenic substrate and the simultancous measuremem of emtttcd light. The substrate in this dctcction system is a luciferin-bascd derivative. Light detectton is performed using a photon-counting camera, and the blotted protcins arc vtsualt:ed as bright spots. This teehnique is very similar to chemiluminescence in scnsittvity and sjH-od of detection but is not as widcly used, and bioluminogenic substrates are commercially availabie. PVDF is the preferred membranę for roiolummescent dctcction because nitrocellulose ■fiembranes may contain substances chat inhibit [ uei erase activity and thus interfere with the assay.

Chemifluorescent Detection Chemifluorescence is the enzymacic conversion of a substrate to a fluorescent product. Fluorogenic compounds (nonfluorescent or weakly fluorescent substances chat can be converted to fluorescent Products) are available to use with a wicie variety of enzymes, including AP and HRP. The enzymc cleaves a phosphate group from a fluorogenic substrare to yield a highly fluorescent product. The fluorescence can be detected using a fluorescence imager such as the Molecular Imager FX~ Pro Plus system or VersaDoc system and ąuantitated using Quantity One* software. Chemifluorescence can provide a stable fluorescent reaction product, so that blots can be scanned at a convenient time. The method is compatible with standard stripping and reprobing procedures.

Fluorescent Detection ln fluorescent detection, the secondary antibody is labeled with a fluorophore such as fluorescein (F1TC), Texas Red, rhodamine (TRITC), or R-phycoerythrin. The main advantage of fluorescent detection is that it can providc a 10-fold greater linear dynamie rangę with only 2—4-fold reduced sensitivity over chemiluminescent detection. Fluorescent western biot detection can therefore provide better linearity and better ąuantitation wtthin the detection limits. Fluorescent detection also allows multiplexing. Multiplexing with

diffcrent colored fluorophores allows simultancous detection of scveral target protcins on thc same biot.

Autoradlography

Radioisotopes can be used to lahel probes (in thc casc of western blots, thc secondary antibodics) for most blotting applications. The radioisotopes commonly used in biological Sciences indudc ^5sS, ^J2P, ⁵ⁱP, ^HC, and ^i:5I. Todetect the radioactivity, thc most widcly used method is autoradlography on X*ray film. Autoradlography provides a good combination of sensitivity and resolution without a largo invcstmcnt. For direct auroradiography without intensilying screens or scintillators, rhe respon.se of thc film is linear only within a rangę of 1-2 order* of magnitude, When intcnsifying screens or ftuomgr.iphic scintillators arc used to incrcase sensitivlty, thc responsc of the film is nonlincar, but it can casfly be madę linear by preexposing the film to a (łasił of light.

Phosphor imagers such as Bio-Rads Molecular Imager FX" family of imagers offer an altemarive to film detection merhods. The initial invcstment in instrumentation offers inereased sensitivity and dynamie rangę compared to X-ray film, and exposure times are 10 to 20 timcs shorter than thosc for film. The ability to accurately quantitatc data is also much greater with storage phosphor screens because the linear dynamie rangę of phosphor imagers is significantly greater, 4.8 orders of magnitude. The advantage of a linear dynamie rangę is that there is a direct relationship between signal intensity and actual quantity. Thts enables accurate quantitation and the elimination of overexposure and saturated signals.

Colorimctric Detection Several substrates are converted to a colored precipitate by enzymes such as HRP or AP, which are usually conjugated to the secondary antibody. The most commonly used substrates include 5-bromo-4-chloro-3'indolyl phosphate/Nitroblue Tetrazolium (BCIP/NBT) for AP and diaminobenzidine (DAB) or 4-chloro*l-naphthol (4CN) for HRP. As the precipitate aceumulares, a visible colored signal develops on the biot. The enzyme reaction can be monitored and stopped when the desired signako> noise level is reached. The method is easier than any

A. Chemlumineocenoo

B. BcJumnoecenca

C. Chemifluorescence

D. Fłuorescence/autoradtograpny

E. Colońmetric detection

Target proten Pnmary antibody

Fig. 1. Mechanismt of detection method chemistries.

In each method of western biot detection. a detectabte signal is generated foBowing binding of an antibody specific for the protein of interest. Chemiluminescent, biolgmlneseent, and chemifluorescent detection all rety on generation of a light signal. In chemiluminascent (At and bfolumineseeoi (B) tieteciipnjhe^ reaction itseif emils light. Chemiluminescent and biduminescent detection are distinguished by the source of the substrate. In chemifluorescent detection (C). the product is fluorescent. Both fluorescent detection and autoradlography (D) record the signal generated by a labeled secondary antibody. In fluorescent detection. the antibody is labeled with a fluorophore. while in autoradiography. it is labeled with a radioastwe isotope. In colorimetric detection (E). the signal is a colored precipitate.

A

t

Secondary antibody Enzyme conjugate

S Substrate P Product ₍

* labdfradciabeior fluorophore) Emitted light or radeicn