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ケーススタディ 2: 感度 (XP®モノクローナル抗体)

XP®モノクローナル抗体の優れた感度

特異性は抗体の評価を担う最も重要な基準ですが、研究サンプルが限られる場合やタンパク質の内在性レベルが低い場合は抗体の感度が重要になります。CSTのXP®モノクローナル抗体である#5558 Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAb#4858 Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAbと競合製品との比較試験では、XP®モノクローナル抗体の優れた感度が実証されています。

#5558 Phospho-GSK-3β (Ser9) (D85E12) XP® Rabbit mAbと競合製品との比較

  • ウェスタンブロッティング (Figure 1) において、#5558は優れた特異性と感度を示しました。
  • #5558は著しく低い抗体濃度で試験しており、製品の感度の高さを証明しています。
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.

(競合製品のシグナルを検出するため、通常より長めに露出しています。)

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#4858 Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAbと競合製品との比較

  • ウェスタンブロッティング (Figure 2) において、#4858は競合製品よりも低濃度で標的タンパク質を検出しました。
  • 使用した抗体濃度が著しく低いにも関わらず標的タンパク質を鮮明に検出していることから、#4858が優れた特異性と感度を示すことがわかります。
  • #4858は様々なアプリケーションでご使用いただけるので、研究の進行に伴うアプリケーションの追加に対応できます。
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.

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#4858 Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAbと他のラビットモノクローナル抗体との比較

  • ウェスタンブロッティング (Figure 3A, 3B) において、CSTの3つすべての抗体は適切な分子量に単一の特異的バンドを検出しました 。これはCSTの高水準の製品規格によるもので、この厳格な品質管理が高品質な製品のご提供を可能にしています。
  • #4858は、#4856#4857に比べて低いライセート濃度でもシグナルを検出しました (Figure 3B)。
  • パラフィン包埋結腸がん組織を用いた免疫組織染色 (Figure 4) では、最適な抗体濃度において#4858#4857よりも高い感度を示しました 。
  • 免疫蛍光細胞染色 (Figure 5A) では、最適な抗体濃度において#4858#4856よりもわずかに強いシグナルを検出しました。
  • ハイコンテントプラットフォームを用いて、段階希釈して免疫蛍光強度を定量化したところ、#4858#4856よりも有意に高い強度を示しました (Figure 5B)。
  • フローサイトメトリー (Figure 6) において、#4858#4856より低い濃度でも強いシグナルを検出でき、薬剤処理による変化をよりはっきりと観察できました (Figure 6)。
  • Figure 3-6の結果より、#4858は他のラビットモノクローナル抗体よりも高感度であることが実証されました。
Comparing western blot analysis using XP<sup>®</sup> #4858 and non-XP<sup>®</sup> #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.

ケーススタディ 2: 感度 (XP®モノクローナル抗体)

Pathways & Diagrams for Epigenetic Regulation

XP®モノクローナル抗体パンフレット

XP®モノクローナル抗体の優れたパフォーマンスを競合製品との比較を交えてご紹介しています。ご使用された先生方の声も掲載!

 

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