Anti-MGMT antibody [EPR4398] - BSA and Azide free

• WB

Lab

Western blot - Anti-MGMT antibody [EPR4398] - BSA and Azide free (AB247735)

This data was developed using ab108989, the same antibody clone in a different buffer formulation.

Western blot : Anti-MGMT antibody [EPR4398] (ab108989) staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab108989 was shown to bind specifically to MGMT. A band was observed at 22 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in MGMT CRISPR-Cas9 edited cell line ab286541. The band observed in the CRISPR-Cas9 edited lysate lane below 22 kDa is likely to represent a truncated form of MGMT. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and MGMT CRISPR-Cas9 edited HCT 116 cell lysates were analysed. First, 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-MGMT antibody [EPR4398] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4398-ab108989'>ab108989</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human MGMT knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-mgmt-knockout-hct116-cell-line-ab286541'>ab286541</a>)

Lane 2:

MGMT CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 22 kDa

false

• IP

Unknown

Immunoprecipitation - Anti-MGMT antibody [EPR4398] - BSA and Azide free (AB247735)

This data was developed using ab108989, the same antibody clone in a different buffer formulation.

ab108989 (purified) at 1/30 immunoprecipitating MGMT in 10 μg MCF7 (Lanes 1 and 2, observed at 23 kDa). Lane 3 - PBS. For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution. Blocking buffer and concentration : 5% NFDM/TBST Dilution buffer and concentration : 5% NFDM/TBST

All lanes:

Immunoprecipitation - Anti-MGMT antibody [EPR4398] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4398-ab108989'>ab108989</a>)

Predicted band size: 22 kDa

false

• WB

Lab

Western blot - Anti-MGMT antibody [EPR4398] - BSA and Azide free (AB247735)

This data was developed using ab108989, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MGMT antibody [EPR4398] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4398-ab108989'>ab108989</a>) at 1/1000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 23 kDa

false

• WB

Lab

Western blot - Anti-MGMT antibody [EPR4398] - BSA and Azide free (AB247735)

This data was developed using ab108989, the same antibody clone in a different buffer formulation.

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

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

Lane 3 : MCF7 cell lysate (20 μg)

Lane 4 : Jurkat cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab108989 observed at 22 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab108989 was shown to specifically react with MGMT when MGMT knockout samples were used. Wild-type and MGMT knockout samples were subjected to SDS-PAGE. ab108989 and ab8245 (loading control to MGMT) were diluted 1/1000 and 1/2000 respectively and 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 1/10 000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-MGMT antibody [EPR4398] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4398-ab108989'>ab108989</a>)

Predicted band size: 22 kDa

false

• WB

Unknown

Western blot - Anti-MGMT antibody [EPR4398] - BSA and Azide free (AB247735)

This data was developed using ab108989, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-MGMT antibody [EPR4398] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-epr4398-ab108989'>ab108989</a>) at 1/1000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 22 kDa

Observed band size: 23 kDa

false