As shown previously [27], western blot analysis of eIF4E correlated with TLK1B protein expression. At the same time, eIF4E expression (determined by both western blot and TMA-IHC) did not correlate with ER, PR or HER-2/neu. Consistent with these results, the lack of correlation of eIF4E (detected by western blot) with ER, PR, and HER-2/neu was previously reported [18, 19].
Our results confirm and extend the results previously described by Yang and colleagues [20]. In their study, which utilized a multi-tumor TMA from TARP http://www.cancer.gov/tarp/, they found eIF4E, VEGF, and cyclin D1 were elevated in breast tumors compared to combined normal tissues [20]. The authors also found that eIF4E levels
were moderately correlated Opaganib mw with VEGF and cyclin D1 expression in breast (Spearman’s rank correlation) [20]. Among the major differences between the two studies: this study focused solely on breast cancer, and included validation of western blot and IHC analysis of the same samples. We also verified coefficients of variance to demonstrate plug-to-plug reproducibility. Furthermore, we examined a broader range of downstream proteins, and included more negative controls. Also, we used the ARIOL imaging system whereas they used ACIS. The strength of these two studies supports the idea that IHC can be used in a TMA format for evaluating critical oncogenic proteins. In comparing western blot to IHC, there are advantages and disadvantages to both procedures. One advantage to western blot, traditionally, is that it has provided a greater CH5424802 molecular weight dynamic range for quantitation than IHC. This is especially true, historically, as IHC has
been semi-quantitative and subject to scoring methods. However, with the wider availability of IHC quantitation systems such as ARIOL, IHC has become more quantitative. This also provides potential standardization between different research institutions. The use of TMAs rather than individual IHCs for each specimen also provides institutions the PJ34 HCl ability to analyze a larger set of specimens at a time using similar staining and quantitation procedures. Another advantage to western blot, however, is that the molecular weights of the proteins can be estimated based on the molecular weight standards that are also resolved on the gel. This is particularly important if the antibodies exhibit non-specific staining. The protein of interest can be isolated from the non-specific staining and quantitated. The best way to overcome the problem of non-specific staining in IHC is to use the most specific antibodies available and to optimize the dilutions of antibodies and other staining conditions. Comparisons of positive and negative controls also help confirm specificity.