Anti-Thymidine Kinase 1/TK1 antibody [EPR3193]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (AB76495)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Thymidine Kinase 1/TK1 with Purified ab76495 at 1/200 dilution (0.1 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488 ,ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Left). Unlabeled control - /.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (AB76495)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling Thymidine Kinase 1/TK1 with purified ab76495 at 1/200 dilution (0.85 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Lab

Immunoprecipitation - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (AB76495)

Purified ab76495 at 1/20 dilution (0.8μg) immunoprecipitating Thymidine Kinase 1/TK1 in MOLT-4 whole cell lysate.
Lane 1 (input) : MOLT-4 (Human lymphoblastic leukemia T lymphoblast)
Lane 2 (+) : ab76495 + MOLT-4 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab76495 in MOLT-4 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 24 kDa

All lanes:

Immunoprecipitation - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (ab76495)

Predicted band size: 25 kDa

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• WB

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Western blot - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (AB76495)

All lanes:

Western blot - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (ab76495) at 1/1000 dilution

Lane 1:

MOLT-4 (Human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 2:

U-2 OS (Human bone osteosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

SW620 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 5:

Human tonsil lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 25 kDa

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• WB

Lab

Western blot - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (AB76495)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : TK1 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : Jurkat whole cell lysate (20 μg)
Lane 4 : MOLT4 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab76495 observed at 25 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76495 was shown to specifically react with TK1 in wildtype HAP1 cells. No band was observed when TK1 knockout samples were examined. Wild-type and TK1 knockout samples were subjected to SDS-PAGE. ab76495 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (ab76495)

Predicted band size: 25 kDa

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• WB

Lab

Western blot - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (AB76495)

Lanes 1- 2 : Merged signal (red and green). Green - ab76495 observed at 25 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab76495 was shown to react with Thymidine Kinase 1/TK1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266230 (knockout cell lysate ab257745) was used. Wild-type HEK-293T and TK1 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab76495 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Thymidine Kinase 1/TK1 antibody [EPR3193] (ab76495) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

TK1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human TK1 (Thymidine Kinase 1) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-tk1-thymidine-kinase-1-knockout-hek-293t-cell-line-ab266230'>ab266230</a>)

Predicted band size: 21 kDa,25 kDa

Observed band size: 21 kDa,25 kDa

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