Anti-MGMT antibody (ab80513)
Key features and details
- Rabbit polyclonal to MGMT
- Suitable for: WB, ICC/IF, IHC-P
- Knockout validated
- Reacts with: Human
- Isotype: IgG
Overview
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Product name
Anti-MGMT antibody
See all MGMT primary antibodies -
Description
Rabbit polyclonal to MGMT -
Host species
Rabbit -
Tested Applications & Species
See all applications and species dataApplication Species ICC/IF HumanIHC-P HumanWB Human
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Immunogen
Synthetic peptide corresponding to Human MGMT aa 1-100 (N terminal) conjugated to keyhole limpet haemocyanin.
(Peptide available asab89380) -
Positive control
- WB: Recombinant Human MGMT protein (ab79251), HeLa, irradiated HeLa, Jurkat, HepG2 and Caco2 whole cell lysates. IHC-P: Human normal tonsil tissue. ICC/IF: Wild-type HAP1 cells.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab80513 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
GuaranteedTested applications are guaranteed to work and covered by our Abpromise guarantee.
PredictedPredicted to work for this combination of applications and species but not guaranteed.
IncompatibleDoes not work for this combination of applications and species.
Application Species ICC/IF HumanIHC-P HumanWB HumanApplication Abreviews Notes WB Use a concentration of 1 µg/ml. Detects a band of approximately 25 kDa (predicted molecular weight: 21 kDa).ICC/IF Use a concentration of 1 µg/ml.IHC-P Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.Notes WB
Use a concentration of 1 µg/ml. Detects a band of approximately 25 kDa (predicted molecular weight: 21 kDa).ICC/IF
Use a concentration of 1 µg/ml.IHC-P
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.Target
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Function
Involved in the cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Repairs alkylated guanine in DNA by stoichiometrically transferring the alkyl group at the O-6 position to a cysteine residue in the enzyme. This is a suicide reaction: the enzyme is irreversibly inactivated. -
Sequence similarities
Belongs to the MGMT family. -
Cellular localization
Nucleus. - Information by UniProt
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Database links
- Entrez Gene: 4255 Human
- Omim: 156569 Human
- SwissProt: P16455 Human
- Unigene: 501522 Human
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Alternative names
- 6 O methylguanine DNA methyltransferase antibody
- 6-O-methylguanine-DNA methyltransferase antibody
- Agat antibody
see all
Images
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Western blot - Anti-MGMT antibody (ab80513)
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 - ab80513 observed at 22 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab80513 was shown to specifically react with MGMT when MGMT knockout samples were used. Wild-type and MGMT knockout samples were subjected to SDS-PAGE. ab80513 and ab8245 (loading control to GAPDH) were diluted 1μg/mL 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 1/10000 dilution for 1 h at room temperature before imaging. -
Immunocytochemistry/ Immunofluorescence - Anti-MGMT antibody (ab80513)ab80513 staining MGMT in wild-type HAP1 cells (top panel) and MGMT knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab80513 at 1μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody (ab80513)IHC image of MGMT staining in Human normal tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab80513, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
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Western blot - Anti-MGMT antibody (ab80513)All lanes : Anti-MGMT antibody (ab80513) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Irradiated HeLa Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 5 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?
Additional bands at: 120 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 4 minutes
Methylated-DNA--protein-cysteine methyltransferase (MGMT) contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.
Protocols
Datasheets and documents
References (0)
ab80513 has not yet been referenced specifically in any publications.
Images
-
Western blot - Anti-MGMT antibody (ab80513)
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 - ab80513 observed at 22 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab80513 was shown to specifically react with MGMT when MGMT knockout samples were used. Wild-type and MGMT knockout samples were subjected to SDS-PAGE. ab80513 and ab8245 (loading control to GAPDH) were diluted 1μg/mL 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 1/10000 dilution for 1 h at room temperature before imaging. -
Immunocytochemistry/ Immunofluorescence - Anti-MGMT antibody (ab80513)
ab80513 staining MGMT in wild-type HAP1 cells (top panel) and MGMT knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab80513 at 1μg/ml concentration and ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
-
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MGMT antibody (ab80513)
IHC image of MGMT staining in Human normal tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab80513, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
-
Western blot - Anti-MGMT antibody (ab80513)All lanes : Anti-MGMT antibody (ab80513) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Irradiated HeLa Whole Cell Lysate
Lane 3 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lane 4 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 5 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 21 kDa
Observed band size: 25 kDa why is the actual band size different from the predicted?
Additional bands at: 120 kDa. We are unsure as to the identity of these extra bands.
Exposure time: 4 minutes
Methylated-DNA--protein-cysteine methyltransferase (MGMT) contains a number of potential phosphorylation sites (SwissProt) which may explain its migration at a higher molecular weight than predicted.
