Anti-MGMT antibody [EPR4397]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody [EPR4397] (AB108630)

Unpurified ab108630, at a 1/250 dilution, staining MGMT in paraffin embedded Human spleen tissue by Immunohistochemistry.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody [EPR4397] (AB108630)

Unpurified ab108630, at a 1/250 dilution, staining MGMT in paraffin embedded Human clear cell carcinoma tissue by Immunohistochemistry.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody [EPR4397] (AB108630)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung cancer tissue sections labeling MGMT with purified ab108630 at 1 : 800 dilution (0.94 μg/ml). Heat mediated antigen retrieval was performed using Citrate buffer, pH 6.0. ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• WB

Lab

Western blot - Anti-MGMT antibody [EPR4397] (AB108630)

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 - unpurified ab108630 observed at 22 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab108630 was shown to specifically react with MGMT when MGMT knockout samples were used. Wild-type and MGMT knockout samples were subjected to SDS-PAGE. ab108630 and ab8245 (loading control to GAPDH) 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.

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Western blot - Anti-MGMT antibody [EPR4397] (ab108630)

Predicted band size: 22 kDa

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• WB

Lab

Western blot - Anti-MGMT antibody [EPR4397] (AB108630)

All lanes:

Western blot - Anti-MGMT antibody [EPR4397] (ab108630) at 1/5000 dilution

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MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 22 kDa

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• WB

Lab

Western blot - Anti-MGMT antibody [EPR4397] (AB108630)

Western blot : Anti-MGMT antibody [EPR4397] (ab108630) 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, ab108630 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 [EPR4397] (ab108630) 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

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• WB

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Western blot - Anti-MGMT antibody [EPR4397] (AB108630)

All lanes:

Western blot - Anti-MGMT antibody [EPR4397] (ab108630) at 1/1000 dilution

Lane 1:

MCF7 cell lysate at 10 µg

Lane 2:

Jurkat ell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

HT29 cell lysate at 10 µg

Predicted band size: 22 kDa

Observed band size: 23 kDa

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• WB

CiteAb

Western blot - Anti-MGMT antibody [EPR4397] (AB108630)

MGMT western blot using anti-MGMT antibody [EPR4397] ab108630. Publication image and figure legend from Lai, S. W., Huang, B. R., et al., 2018, Int J Mol Sci, PubMed 29301329.

ab108630 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab108630 please see the product overview.

Effects of TMZ treatment on MGMT and Cx43 expression in GBM. Human U251 glioma cells were treated with TMZ or non-treated to induce TMZ-resistant U251 cells and parental U251 cells, respectively. (A) The TMZ-resistant cells and the parental cells have a distinctive morphology. Scale bar = 20 μM. (B) Determination of the TMZ (0–1000 μM, for 72 h)-induced cell death in parental U251 cells and TMZ-resistant U251 cells. Analyzed by SRB assay for cell viability. Quantitative data are presented as mean ± SEM of three independent experiments. * p < 0.05 compared with the parental group; (C) MGMT, Cx43, and p-Cx43 expression were determined using Western blot analysis in parental and TMZ-resistant cells.

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