Digital image analysis for rapid quantification of total RNA and cDNA for SMART-PCR.

when duplicate blots were probed with the avidin-peroxidase conjugate (Figure 1B, lanes 5′–8′) ExtrAvidin® (Sigma, St. Louis, MO, USA). Unlike the secondary antibody conjugate used in Figure 1A, the avidin-peroxidase conjugate used in Figure 1B did not recognize the electrophoresed immunoglobulins within the immunoprecipitate. Moreover, specific bands of ezrin were readily detected in immunoprecipitates using biotinylated ezrin antibody and the ExtrAvidin-peroxidase, regardless of whether samples were reduced or not (Figure 1B, lanes 6 and 8). Comparison of serial dilutions of total placental microvilli revealed that biotinylation of the affinity-purified ezrin antibody had little or no detectable effect on its sensitivity or specificity (compare Figure 1A, lanes 1–4, and Figure 1B, lanes 1–4). Moreover, we have found that the sensitivity and specificity of more than a half dozen other biotinylated rabbit polyclonal antibodies were retained on blots. Because a wide range of biotinylated antibodies from different species is available through commercial suppliers, we speculate that biotin-labeling will also be applicable to many monoclonal antibodies. However, the effect of biotinylation on antibody sensitivity or specificity must be determined empirically for each immunoreagent. Table 1 describes a procedure for biotinylation. Although ezrin was detected in immunoprecipitates electrophoresed under reducing conditions using the conventional method for blotting, the considerable amount of background staining would preclude visualization of many other bands, especially in the region of the IgG-heavy chain (Figure 1A, asterisk). However, using the improved method for blotting, it is possible to detect polypeptides even when they co-migrate with the IgG-heavy chain (4). Thus, our improved method considerably expands the number of different-sized bands that can be visualized on immunoblots of immunoprecipitates. In addition, this method is simple, rapid, economical, and requires only small quantities of antibody.