Anti-MGMT antibody [MT3.1] - BSA and Azide free

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody [MT3.1] - BSA and Azide free (AB255966)

This data was produced using the same clone but in a different formulation, ab39253.

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma tissue labeling MGMT with ab39253 at 1/200 followed by a ready to use secondary. Positive staining on human prostate carcinoma. The section was incubated with ab39253 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use secondary. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody [MT3.1] - BSA and Azide free (AB255966)

This data was produced using the same clone but in a different formulation, ab39253.

Immunohistochemical analysis of paraffin-embedded human lung carcinoma tissue labeling MGMT with ab39253 at 1/200 followed by a ready to use secondary. Positive staining on human lung carcinoma. The section was incubated with ab39253 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Secondary antibody only control : Secondary antibody is a ready to use secondary. Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• WB

Lab

Western blot - Anti-MGMT antibody [MT3.1] - BSA and Azide free (AB255966)

This data was produced using the same clone but in a different formulation, ab39253.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MGMT antibody [MT3.1] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-mt31-ab39253'>ab39253</a>) at 1/500 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

HeLa (human cervix adenocarcinoma epithelial cell ), whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

false

Exposure time: 92s

• WB

Lab

Western blot - Anti-MGMT antibody [MT3.1] - BSA and Azide free (AB255966)

This data was produced using the same clone but in a different formulation, ab39253.

All lanes:

Western blot - Anti-MGMT antibody [MT3.1] - BSA and Azide free (ab255966) at 1/500 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 20 µg

Lane 2:

MGMT knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell) at 20 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte) at 20 µg

Predicted band size: 22 kDa

false

• WB

Lab

Western blot - Anti-MGMT antibody [MT3.1] - BSA and Azide free (AB255966)

This data was developed using ab39253, the same antibody clone in a different buffer formulation.

Western blot : Anti-MGMT antibody [MT3.1] (ab39253) staining at 1/500 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab39253 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 knockout cell line ab286541. The band observed in the knockout 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 knockout 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-Mouse IgG H&L 800CW and Goat anti-Rabbit IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-MGMT antibody [MT3.1] (<a href='/en-us/products/primary-antibodies/mgmt-antibody-mt31-ab39253'>ab39253</a>) at 1/500 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 knockout HCT 116 cell lysate at 20 µg

Secondary

Lanes 1 - 2:

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

Lanes 1 - 2:

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

Observed band size: 22 kDa

false