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ケーススタディ 1: 特異性 (XP®モノクローナル抗体)


活性型特異的抗体 (特にリン酸化抗体) は、近似の配列を保持する関連タンパク質とも交差し、非特異的バンドや誤ったシグナルを検出する可能性があります。CSTのリン酸化XP®モノクローナル抗体である#4370 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb#3077 Phospho-Met (Tyr1234/1235) (D26) 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特異性が実証されています。

#4370 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) 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、#4370が最もバックグラウンドが低く特異的な強いシグナルを検出しました。免疫蛍光細胞染色 (Figure 2) においても、#4370は優れた特異性を示しました。
  • Figure 1とFigure 2の結果より、Competitor 1の抗体は標的タンパク質以外とも交差し、細胞染色の結果が誤ったシグナルに基づく可能性が示されました。それに対し、#4370は標的タンパク質を特異的に検出するため信頼ある細胞染色の結果を示しました。
  • 免疫組織染色 (Figure 3) においても、#4370は優れた特異性を示しました。Competitor 2の抗体は希釈率によらず標的タンパク質の染色を判別しづらいのに対し、#4370はバックグラウンドが低く特異的で強い染色を示しました。
  • 様々なチロシンリン酸化組換えタンパク質を用いたウェスタンブロッティング (Figure 4) において、#4370は標的タンパク質以外のリン酸化タンパク質と交差せず、優れた特異性が実証されました。
  • #4370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 #4370 Competitor 1 Competitor 2
Western Blot Dilution 1:2000 1:100 1:100
Assay Concentration (µg/mL) 0.222 1 10
Recommended Applications W, IP, IHC-P, IF-IC, F W, IP, IF W, IHC-P, IHC-F
Species Cross-reactivity H, M, R, Hm, Mk, Mi, Dm, Z, B, Dg, Pg, Sc, (Ce) H, M, R, Dg H, M, R, (C, X, Z)
Side by side western blot analysis of XP<sup>®</sup> #4370 vs competitors

Figure 1. Jurkat cells were either left untreated or treated with the phorbol ester TPA #4174 (200 nM,10 min), following overnight serum-deprivation, to induce phosphorylation of p44/42 protein. #4370 was used at the recommended dilution and both competitor antibodies were used at the highest concentration of the manufacturer’s recommended dilution range. All antibodies showed the expected signal induction upon TPA treatment. Both competitor antibodies showed greater background staining, with competitor 1 showing a number of cross-reacting bands and competitor 2 also showing weaker overall staining.

Side by side immunofluorescent comparison of XP<sup>®</sup> #4370 vs competitors

Figure 2. HeLa cells were treated with the MEK 1/2 inhibitor U0126 #9903 (10 µM, 2 hr) or treated with TPA #4174 (200 nM, 30 min), following overnight serum deprivation. #4370 was used at the optimal IF-IC recommended dilution of 1:200 and competitor 1 antibody was tested at a dilution range of 1:50-1:500 per the manufacturer’s suggestion (data not shown). At the concentration determined optimal, the competitor antibody exhibited more background staining, both nuclear and cytoplasmic, in the U0126-treated cells and only weak, diffuse cytoplasmic staining in the stimulated cells. The expected fold-induction following TPA treatment was observed with #4370, but very little induction was observed in the competitor-stained cells and the antibody appeared “dirtier” overall.

Side by side immunohistochemical assays comparison of XP<sup>®</sup> #4370 vs comptetitors

Figure 3. #4370 was compared to a competitor’s IHC-approved antibody tested at two concentrations. IHC analysis was performed on #8103 [paraffin-embedded NIH/3T3 cell pellets, treated with either TPA #4174 (upper) or U0126 #9903 (middle)] and paraffin-embedded human ovarian carcinoma (lower). At the optimal recommended dilution of 1:400, #4370 showed the appropriate staining in the cell pellet system. The competitor 2 antibody required a 1:400 dilution to eliminate staining in the negative control U0126-treated cells. However, signal in the positive control TPA-treated cells was considerably reduced at this dilution. At 1:100 (lowest suggested dilution), the competitor 2 antibody staining was weak in human ovarian carcinoma tissue and cannot be considered specific given the staining observed in U0126-treated cells. Tissue staining was barely present at a 1:400 dilution of the competitor 2 antibody. In contrast, #4370 demonstrated strong specific staining in human ovarian carcinoma.

Side by side western blot analysis using a panel of recombinant tyrosine-phosphorylated proteins

Figure 4. Western blot analysis using a panel of recombinant tyrosine-phosphorylated proteins shows no detectable cross-reactivity using #4370, and significant cross-reactivity with other tyrosine phosphorylated proteins using both competitor antibodies tested. Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 was used to demonstrate protein loading and verify molecular weight of the tagged recombinant proteins. These results demonstrate that #4370 displays exceptional specificity.


#3077 Phospho-Met (Tyr1234/1235) (D26) 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 5) において、#3077は適切な分子量に単一の特異的バンドを検出しましたが、競合製品は標的タンパク質以外も検出しました。
  • 免疫組織染色 (Figure 6) において、#3077は様々なRTK阻害剤や刺激剤で処理したパラフィン包埋細胞ペレットで特異性を確認できましたが、競合製品は確認できませんでした。
  • HCC827 xenograftを用いた免疫組織染色 (Figure 7) では、#3077と競合製品ともに特異的な染色が見られました。
  • ホスファターゼ未処理と処理済のパラフィン包埋肺がん組織を用いた免疫組織染色 (Figure 8) においても、#3077の特異性を確認できました。
  • Figure 5-8の結果より、競合製品は標的タンパク質以外とも交差し免疫組織染色の結果が誤ったシグナルに基づく可能性が示されました。それに対し、#3077は標的タンパク質を特異的に検出するため信頼ある免疫組織染色の結果を示し、優れた特異性が実証されました。
Extracts treated with growth factors vs Competitor

Figure 5. A single band at 145 kDa was observed by western blotting in HGF-stimulated, but not in unstimulated A431 cells using #3077 (upper). Extracts treated with growth factors that activate other RTKs or that overexpress other RTKs or cytoplasmic tyrosine kinases were negative. By comparison, the competitor phospho-Met antibody recognizes several non-specific bands (lower). Both membranes were developed on the same film with the same exposure time (10 seconds).

Immunohistochemical analysis of paraffin-embedded MKN45 cells

Figure 6. Immunohistochemical analysis of paraffin-embedded MKN45 cells treated with the Met inhibitor SU11274 showed no staining as compared to the control using both #3077 and the competitor’s antibody. EGF treatment and HRG treatment of MDA-MB-468 and T47D breast carcinoma cell lines, respectively, showed no increase in staining compared to control using #3077. In contrast, the competitor’s antibody detected non-specific staining with both treatments, indicating cross-reactivity with other RTKs.

Cell Signaling Technology #3077 HCC827 xenograft vs Competitor

Figure 7. Immunohistochemical analysis of paraffin-embedded HCC827 xenograft comparing #3077 (left) and a competitor’s antibody (right) gives the appearance of specific staining for both products.

Phosphatase treatment of paraffin-embedded human lung carcinoma using #3077

Figure 8. Phosphatase treatment of paraffin-embedded human lung carcinoma confirms phospho-specificity of #3077.

ケーススタディ 1: 特異性 (XP®モノクローナル抗体)

Pathways & Diagrams for Epigenetic Regulation