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While specificity is the most important criterion for a great antibody, sensitivity can become important when research samples are limited or a protein is expressed at low endogenous levels. We compared Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb #5558, and Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 with competitor products using western blot analysis. We also compared #4858 with other Cell Signaling Technology rabbit monoclonal antibodies #4856 and #4857.

XP® Sensitivity Comparison featuring Phospho-GSK-3β #5558

  • 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.
  CST #5558 Competitor 1 Competitor 2
Western Blot Dilution 1:1000 1:200 1:500, 1:250
Assay Concentration (µg/mL) 0.29 0.5 1
Recommended Applications W, IP, IF-IC, F W, IP W, IF-IC, F
Species Cross-reactivity H, M, R, Hm H, M H
Western blot analysis of CST #5558 against competitors

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.

XP® Sensitivity Comparison featuring Phospho-S6 #4858

  • 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.
  CST #4858 Competitor 1
Western Blot Dilution 1:2000 1:1000
Assay Concentration (µg/mL) 0.029 Unknown
Recommended Applications W, IHC-P, IHC-F, IF-IC, F W
Species Cross-reactivity H, M, R, Mk, Sc, (C) H, (R)
Western blot analysis of CST #4858 against competitors

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).
Comparing western blot analysis using XP® #4858 and non-XP® #4856 and #4857

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.

Immunohistochemical analysis of paraffin-embedded colon carcinoma.

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.

Confocal immunofluorescent analysis of C6 cells, LY294002, U0126.

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.

Side by side immunofluorescent titration analysis.
 
Flow cytometric titration analysis of Jurkat cells

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.

Flow cytometric analysis of Jurkat cells.