重组Anti-Thymidine Phosphorylase抗体[EPR25629-129]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A431 (Human epidermoid carcinoma epithelial cell) cells labelling Thymidine phosphorylase with ab284861 at 1/500 dilution (0.1ug)/(Red) compared with a Rabbit monoclonal IgG (ab172730)/(Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody)/(Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling Thymidine phosphorylase with ab284861 at 1/5000 (0.103 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab284861 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A431 cells labelling Thymidine phosphorylase with ab284861 at 1/50 (10.34 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in A431 cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling Thymidine phosphorylase with ab284861 at 1/50 (10.34 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in THP-1 cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Immunohistochemical analysis of paraffin-embedded Human ovarian cancer tissue labelling Thymidine phosphorylase with ab284861 at 1/5000 (0.103 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human ovarian cancer. The section was incubated with ab284861 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling Thymidine phosphorylase with ab284861 at 1/5000 (0.103 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the interstitial cells of human colon (PMID : 16361565). The section was incubated with ab284861 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IP

Lab

Immunoprecipitation - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Thymidine phosphorylase was immunoprecipitated from 0.35 mg A431 (human epidermoid carcinoma epithelial cell) whole cell lysate 10 ug with ab284861 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab284861 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : A431 (human epidermoid carcinoma epithelial cell) whole cell lysate 10 ug

Lane 2 : ab284861 IP in A431 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab284861 in A431 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (ab284861)

Predicted band size: 50 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 137 seconds

All lanes:

Western blot - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (ab284861) at 1/1000 dilution

All lanes:

A431 (human epidermoid carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

VeriBlot for IP secondary antibody(HRP)(<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Low expression : human ovary (PMID : 2328255).

The bands nearby 25 kDa could be non-specific bands.

Exposure time : Lane 1-3 : 3.25 seconds Lane 4-5 : 37 seconds

All lanes:

Western blot - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (ab284861) at 1/1000 dilution

Lane 1:

Human tonsil tissue lysate at 20 µg

Lane 2:

human ovary cancer tissue lysate at 20 µg

Lane 3:

human ovary tissue lysate at 20 µg

Lane 4:

human colon tissue lysate at 20 µg

Lane 5:

human liver tissue lysate at 20 µg

Secondary

All lanes:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

false

• WB

Lab

Western blot - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (AB284861)

Western blot : Anti-Thymidine Phosphorylase antibody [EPR25629-129] ab284861 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 50 kDa in Wild-type HeLa cell lysates with no signal observed at this size in TYMP knockout HeLa cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Thymidine Phosphorylase antibody [EPR25629-129] (ab284861) at 1/1000 dilution

Lane 1:

Wild-type HeLa at 20 µg

Lane 2:

Western blot - Human TYMP (Thymidine Phosphorylase) knockout HeLa cell line (<a href='/products/cell-lines/human-tymp-thymidine-phosphorylase-knockout-hela-cell-line-ab265138'>ab265138</a>) at 20 µg

Lane 3:

THP-1 at 20 µg

Lane 4:

Jurkat at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

false