TYMP Antibody / Thymidine phosphorylase / PD-ECGF (FY13337)

Immunoprecipitating PD-ECGF/TYMP in SiHa whole cell lysate. Western blot analysis of PD-ECGF/TYMP using anti-TYMP antibody. Lane 1: SiHa whole cell lysates (30ug), Lane 2: Rabbit control IgG instead of anti-TYMP antibody in SiHa whole cell lysate, Lane 3: anti-TYMP antibody (2ug) + SiHa whole cell lysate (500ug). After electrophoresis, proteins were transferred to a membrane. Then the membrane was incubated with rabbit anti-TYMP antibody at a dilution of 0.5 ug/ml and probed with a goat anti-rabbit IgG-HRP secondary antibody. The signal is developed using ECL Plus Western Blotting Substrate. The input lane shows the characteristic doublet at approximately 50 kDa and in the high 40 kDa region. In contrast, the immunoprecipitated material displays a single band at approximately 50 kDa, indicating selective enrichment of the full length TYMP species. The control IgG lane shows no detectable signal, confirming specificity of the immunoprecipitation.

Western blot analysis of PD-ECGF/TYMP using anti-TYMP antibody. Electrophoresis was performed on a 10% SDS-PAGE gel at 80V (Stacking gel) / 120V (Resolving gel) for 2 hours. Lane 1: human whole cell lysates, Lane 2: human SiHa whole cell lysates, Lane 3: human THP-1 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-TYMP antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using an ECL Plus Western Blotting Substrate. A predominant band is detected at approximately 50 kDa in all samples, slightly below the predicted ~55 kDa molecular weight but consistent with the known migration behavior of thymidine phosphorylase. A weaker lower band is also observed in the high 40 kDa range, which likely represents a processed or partially cleaved form of TYMP rather than a distinct isoform.

Flow Cytometry analysis of cells using anti-TYMP antibody. Overlay histogram showing cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-TYMP antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

Flow Cytometry analysis of SiHa cells using anti-TYMP antibody. Overlay histogram showing SiHa cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-TYMP antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.