Anti-MTH1 antibody [EPR15934]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-MTH1 antibody [EPR15934] (AB197028)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MTH1 antibody [EPR15934] (AB197028)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MTH1 antibody [EPR15934] (AB197028)

Intracellular 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 Fluorr^® 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.

• WB

Lab

Western blot - Anti-MTH1 antibody [EPR15934] (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.

All lanes:

Western blot - Anti-MTH1 antibody [EPR15934] (ab197028) at 1/2000 dilution

Lane 1:

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

Lane 2:

NUDT1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human NUDT1 (MTH1) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-nudt1-mth1-knockout-hek-293t-cell-line-ab266400'>ab266400</a>)

Lane 3:

HAP1 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 23 kDa

Observed band size: 18 kDa

false

• WB

Unknown

Western blot - Anti-MTH1 antibody [EPR15934] (AB197028)

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

Blocking/dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MTH1 antibody [EPR15934] (ab197028) at 1/2000 dilution

Lane 1:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg

Lane 2:

A549 (Human lung carcinoma) whole cell lysate at 20 µg

Lane 3:

Human fetal thymus lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 23 kDa

Observed band size: 18 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-MTH1 antibody [EPR15934] (AB197028)

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:

Western blot - Anti-MTH1 antibody [EPR15934] (ab197028)

Predicted band size: 23 kDa

false