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?

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.

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.

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.

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.

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

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.