Case Study 2: eXceptional Sensitivity of XP® Monoclonal Antibodies
Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 Compared to Other Rabbit Monoclonal Antibodies
Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 exemplifies the exceptional sensitivity of XP antibodies from Cell Signaling Technology (CST). We compared #4858 to two other rabbit monoclonal antibodies, #4857 and #4856, generated using conventional technologies. At CST, the observation of a specific signal of the appropriate molecular weight by western is the first and most critical requirement for antibody validation in all applications. As shown in Figure 1A and B, all three CST antibodies show a single and specific band of the appropriate molecular weight, consistent with CST’s high standard validation requirements and promise of the highest quality.
The following observations demonstrate superior sensitivity of XMT technology-derived #4858:
- A serial antibody dilution by western demonstrated that XMT technology derived #4858 can detect target-specific signal at lower concentrations compared to #4856, indicating #4858 has superior sensitivity and affinity (Figure 1A).
- Selling stocks of #4858 detect lower lysate concentrations than #4856 or #4857 (Figure 1B).
- 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 2).
- Using confocal immunofluorescent analysis at matched concentrations #4858 appeared to generate a slightly brighter signal (Figure 3A).
- Quantification of immunofluorescence intensity in a serial dilution on a high content platform revealed greater intensity of #4858 compared to #4856 (Figure 3).
- 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 4).
In conclusion, CST’s Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 has the exceptional sensitivity necessary for superior performance in imaging-based assays. Moreover, #4858 passed our performance standards in all applications. Table 1 summarizes our observations.
Figure 1A. 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 1B. Western blot analysis of a serial dilution of extracts from insulin-treated HeLa cells (100 nM for 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 2. 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 3A. Confocal immunofluorescent analysis of C6 cells, LY294002, U0126 and rapamycin-treated for 2 hours (upper) or insulin-treated (100 nM for 30 min., lower), using #4858 (left) or #4856 (right). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5® (fluorescent DNA dye). At optimal concentration (0.25 μg/ml) #4858 appears to generate a stronger signal. For numerical values see panel Figure 3B.
Figure 3B. Side by side immunofluorescent titration analysis comparing #4858 with #4856. Optimal concentration for both antibodies was determined to be 0.25 μg/ml for both antibodies. At this concentration signal brightness was 2 fold higher in #4858 than #4856, demonstrating greater sensitivity.
Figure 4A. Flow cytometric titration analysis of Jurkat cells, LY294002, wortmannin and U0126-treated, comparing #4858 and #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 than #4856. Moreover, fold induction over control was also nearly 2 fold higher (not shown). Taken together, these results indicate greater sensitivity by flow cytometry.
Figure 4B. Flow cytometric analysis of Jurkat cells, untreated (grey fill) or treated with LY294002, wortmannin and U0126 (no fill), comparing #4858 and #4856 at optimal concentration (0.25 and 0.5 μg/ml).
| No. | Antibody | Reactivity | WB | IHC | Flow | IF |
|---|---|---|---|---|---|---|
| 4858 | Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb | H, M, R, Mk, Sc, (C) | ++++ | ++++ | ++++ | ++++ |
| 4856 | Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mAb | H, M, R, Mk | ++ | - | ++ | ++ |
| 4857 | Phospho-S6 Ribosomal Protein (Ser235/236) (91B2) Rabbit mAb | H, M, R | ++ | +++ | N/T | ++ |
Table 1. Summary of Phospho-S6 antibodies compared in the study.
Applications Key: WB=Western Blotting IHC=Immunohistochemistry Flow=Flow Cytometry IF=Immunofluorescence
Testing Data Key: (++++)=Very Highly Recommended (+++)=Highly Recommended (++)=Recommended (-)=Not Recommended N/T=Not Tested
Reactivity Key: H=Human M=Mouse R=Rat Mk=Monkey C=Chicken Sc=S.cerevisiae
Species enclosed in parentheses are predicted to react based on 100% sequence homology.
