MA5-13506

antibody from Invitrogen Antibodies

Targeting: MGMT

Antibody data

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Validation data

Product number: MA5-13506 - Provider product page
Provider: Invitrogen Antibodies
Product name: MGMT Monoclonal Antibody (MT 3.1)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA5-13506 targets MGMT (O-methylguanine-DNA methyltransferase) in FACS, ICC/IF, IHC (P), and WB applications and shows reactivity with Human samples.
Antibody clone number: MT 3.1
Concentration: 0.4 mg/mL

MGMT protein structure - MA5-13506 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of MGMT (O-methylguanine-DNA methyltransferase) using MGMT (O-methylguanine-DNA methyltransferase) Monoclonal Antibody (Product # MA5-13506) on Raji Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Methylated-DNA-protein-cysteine methyltransferase was achieved by transfecting HeLa with Methylated-DNA-protein-cysteine methyltransferase specific siRNAs (Silencer® select Product # s8752, s8750). Western Blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Methylated-DNA-protein-cysteine methyltransferase knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with MGMT Monoclonal Antibody (MT 3.1) (Product # MA5-13506, 1:50 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Methylated-DNA--protein-cysteine methyltransferase.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot was performed using Anti-MGMT Monoclonal Antibody (MT 3.1) (Product # MA5-13506) and a 22 kDa band corresponding to Methylated-DNA-protein-cysteine methyltransferase was observed across positive cell lines (HeLa, MCF7 and Jurkat); and not in negative cell lines (MDA-MB-231 and U-87 MG) tested. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), MDA-MB-231 (Lane 2), MCF7 (Lane 3), Jurkat (Lane 4) and U-87 MG (Lane 5) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0341BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:50 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of MGMT (O-methylguanine-DNA methyltransferase) (green) showing staining in the nucleus and cytoplasm of MCF-7 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a MGMT monoclonal antibody (Product # MA5-13506) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Methylated-DNA-protein-cysteine methyltransferase was performed using 70% confluent log phase MCF7 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with MGMT Monoclonal Antibody (MT 3.1) (Product # MA5-13506) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing nucleus and cytoplasm localization. Panel e represents MGMT (Negative model). Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.