Exceptional Sensitivity of XP^® Monoclonal Antibodies

• Western blot analysis (Figure 1) demonstrates the superior specificity and sensitivity of #5558.
• The concentration of the antibody employed is significantly lower for #5558, further illustrating the sensitivity of the product.

Figure 1. Jurkat cells (left) were either treated with the PI3 Kinase inhibitor LY294002 #9901, which inhibits GSK-3β phosphorylation, or with Calyculin A #9902, which artificially increases phosphorylation levels by inhibiting phosphatases present in cell extracts. For all antibodies, a signal at the appropriate molecular weight (46 kDa) was detected in the calyculin A-treated sample, however, the signal was strongest with #5558. Note: Competitor 2 also shows a significant number of non-specific bands with greater intensity than the appropriate 46 kDa band. Jurkat cells (right) were either left untreated or treated with the apoptosis-inducing reagent, etoposide, a treatment which is known to reduce phosphorylation of GSK-3β. Neither of the competitor products detect phospho-GSK-3β in either the untreated or the etoposide-treated samples.

• Western blot analysis (Figure 2) demonstrates that #4858 detects protein at lower concentrations than the competing product.
• Due to the high level of specificity and sensitivity of #4858, this antibody displays exceptional performance in a broad range of research applications.

Figure 2. Serial dilutions of extracts from untreated or insulin-treated NIH/3T3 cells (100 nM, 10 min) were detected with #4858 at 1:2000 dilution and the lowest dilution within the manufacturer’s recommended range for the competitor antibody (1:1000). The stronger signal was observed using #4858, with the signal of the competitor antibody barely visible in the lanes with lowest amounts of extract or the untreated lane (which contains basal levels of phospho-S6). Note: The competitor antibody displays cross-reacting bands that are stronger than the band of appropriate molecular weight, showing the lack of specificity of this product.

Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP^® Rabbit mAb #4858 compared to Rabbit Monoclonals #4856 and #4857

• As shown in Figures 3A and 3B, all three CST antibodies show a single and specific band at the appropriate molecular weight, consistent with CST’s high standard validation requirements and promise of the highest quality.
• Selling stocks of #4858 detect lower lysate concentrations than #4856 or #4857 (Figure 3B).
• Side by side comparison by IHC on paraffin-embedded colon carcinoma shows stronger signal at matched antibody concentrations, indicating that #4858 is also more sensitive by IHC than #4857 (Figure 4).
• In confocal immunofluorescent analysis at matched concentrations, #4858 appeared to generate a slightly brighter signal (Figure 5A).
• Quantification of immunofluorescence intensity in a serial dilution on a high content platform revealed greater intensity of #4858 compared to #4856 (Figure 5B).
• In a side by side comparison using flow cytometry, greater fluorescence intensity and a greater induction over control at optimal concentration was observed with #4858. Moreover, recommended optimal assay concentration of #4858 is lower than #4856 (Figure 6).

Figure 3A. Western blot analysis of extracts from insulin-treated HeLa cells using serial antibody dilutions of #4858 and #4856, starting at 1 μg/ml. Both antibodies generate a clean and specific signal. However, a much more intense signal was observed with #4858. On the 5 second exposure shown, #4858 generates a strong signal at 1 ng/ml, while the signal from #4856 is much weaker.

Figure 3B. Western blot analysis of a serial dilution of extracts from insulin-treated HeLa cells (100 nM, 10 min) detected with CST selling stocks of #4858, #4856 and #4857 at 1:1000 dilution. Again, the strongest signal was observed using #4858.

Figure 4. Immunohistochemical analysis of paraffin-embedded colon carcinoma using #4858 and #4857 at matched concentrations (80 ng/ml). While both antibodies generate a specific signal, dramatically greater signal strength was observed using #4858.

Figure 5A. Confocal immunofluorescent analysis of C6 cells, LY294002 #9901, U0126 #9903, and rapamycin-treated for 2 hours (upper) or insulin-treated (100 nM, 30 min; lower), using #4858 (left) or #4856 (right). Actin filaments were labeled with Alexa Fluor^® 555 phalloidin (red). Blue pseudocolor = DRAQ5 #4084 (fluorescent DNA dye). At optimal concentration (0.25 μg/ml), #4858 appears to generate a stronger signal. For numerical values see Figure 5B.

Figure 5B. Side by side immunofluorescent titration analysis comparing #4858 with #4856. Optimal concentration for both antibodies was determined to be 0.25 μg/ml. At this concentration signal brightness was 2 fold higher with #4858, demonstrating greater sensitivity.

Figure 6A. Flow cytometric analysis of Jurkat cells, untreated (grey fill) or treated with LY294002 #9901, wortmannin #9951, and U0126 #9903 (no fill), using #4858 (left) and #4856 (right) at optimal concentration (0.25 and 0.5 μg/ml, respectively).

Figure 6B. Side by side immunofluorescent titration analysis comparing #4858 with #4856. Optimal concentration was determined as 0.25 and 0.5 μg/ml, for #4858 and #4856, respectively. At these concentrations signal brightness was almost 2 fold higher using #4858. Moreover, fold induction over control was also nearly 2 fold higher (not shown). These results indicate greater sensitivity of #4858 by flow cytometry.