All lanes : Anti-MTH1 antibody [EPR15934] (ab197028) at 1/2000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : NUDT1 knockout HEK-293T cell lysate
Lane 3 : HAP1 cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 23 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab197028).

Lanes 1- 4: Merged signal (red and green). Green - ab197028 observed at 18 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab197028 was shown to react with MTH1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266400 (knockout cell lysate ab257565) was used. Wild-type HEK-293T and NUDT1 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. ab197028 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 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.

This data was developed using ab197028, the same antibody clone in a different buffer formulation.Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling MTH1 with ab197028 at 1/100 dilution followed by ab97051 Goat Anti-Rabbit IgG H&L (HRP) at 1/500 dilution. Cytoplasm and nuclear staining on Human spleen is observed. Counter stained with Hematoxylin. Negative control: Used PBS instead of primary antibody. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

This data was developed using ab197028, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized PC-3 cells (Human prostate cancer cell line) labeling MTH1 with ab197028 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Cytoplasm and nuclear staining on PC-3 cell line is observed. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows:-
-ve control 1 - ab197028 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2. - ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

This data was developed using ab197028, the same antibody clone in a different buffer formulation.Flow Cytometry analysis of PC-3 (human prostate adenocarcinoma) cells labeling MTH1 with purified ab188474 at 1/230 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077)(1/2000 dilution) was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

This data was developed using ab197028, the same antibody clone in a different buffer formulation.

Lane 1: Wild-type HAP1 cell lysate (20 µg)

Lane 2: MTH1 knockout HAP1 cell lysate (20 µg)

Lane 3: Jurkat cell lysate(20 µg)

Lane 4: A549 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab197028 observed at 18 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab197028 was shown to specifically react with MTH1 when MTH1 knockout samples were used. Wild-type and MTH1 knockout samples were subjected to SDS-PAGE. ab197028 at a dilution of 1/2000 and ab8245 (loading control to GAPDH) diluted at 1/10000 were incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-MTH1 antibody [EPR15934] (ab197028) at 1/2000 dilution

Lane 1 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 2 : A549 (Human lung carcinoma) whole cell lysate
Lane 3 : Human fetal thymus lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Developed using the ECL technique.

Predicted band size: 23 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

This data was developed using ab197028, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

The expression profile observed is consistent with what has been described in the literature (PMID: 11296483). The observed band may be isoform p18.