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Figure 1 Western Blot Validation in Human MOLT4 Cell Lysate with the absence (A) or presence (B) of blocking peptideLoading: 15 &956,g of lysates per lane.Antibodies: Survivin 2235 (1 &956,g/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. |
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Figure 4 Immunocytochemistry Validation of Survivin in MOLT4 CellsImmunocytochemical analysis of MOLT4 cells using anti-Survivin antibody (2235) at 10 &956,g/ml. Cells was fixed with formaldehyde and blocked with 10% serum for 1 h at RT, antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4&730,C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin. |
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Figure 5 Immunofluorescence Validation of Survivin in MOLT4 CellsImmunofluorescent analysis of 4% paraformaldehyde-fixed MOLT4 cells labeling Survivin with 2235 at 10 &956,g/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (green). |
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Figure 2 Independent Antibody Validation (IAV) via Protein Expression Profile in Human Cell LinesLoading: 15 &956,g of lysates per lane.Antibodies: Survivin 2233 (5 &956,g/mL), Survivin 2235 (4 &956,g/mL) and beta-actin 3779 (1 &956,g/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. |
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Figure 3 Western Blot Validation in Human Cell LinesLoading: 15 &956,g of lysates per lane.Antibodies: Survivin 2235 (4 &956,g/mL), 1h incubation at RT in 5% NFDM/TBST.Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. |
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Figure 6 Slot Blot Validation of Survivin in Hepatocellular Carcinoma (HCC) Patients (Zhang et al., 2007) Slot blot analysis of Survivin recombinant protein expression with anti-Survivin antibodies (2235) in the four representative HCC sera. Survivin (Lane 7) was detected in the fourth HCC serum (E), but not in the normal human serum (A) and the other three HCC sera (B-D). Phosphate bufferedsaline (PBS) (Lanes 9 and 10) was used as a negative control.. |
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Figure 7 Western Blot Validation of Survivin with Recombinant Protein (Megliorino et al., 2005)Lane 1: A 28 kDa peptide that corresponded to the size of ORF of survivin was detected in SDS-PAGE with Coomassie blue staining. Lane 2: WB analysis of Survivin recombinant protein expression with anti-Survivin antibodies (2235). |
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Figure 8 Western Blot Validation of Survivin with Recombinant Protein in Sera from Cancer Patients (Megliorino et al., 2005)WB analysis of Survivin recombinant protein expression with anti-Survivin antibodies (2235) in six cancer sera. 6 x His-tagged Survivin recombinant protein (Lanes 4-9) was strongly detected at 28kD, but not in the normal human serum (Lane 3). |