MGMT Antibody (MT 23.2) - BSA Free

Novus Biologicals | Catalog # NB100-168

Novus Biologicals
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Key Product Details

Species Reactivity

Validated:

Human, Mouse (Negative)

Cited:

Human, Mouse

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Flow Cytometry, Flow (Intracellular), Immunocytochemistry/ Immunofluorescence, Simple Western, CyTOF-ready

Cited:

Immunohistochemistry-Paraffin, Western Blot, IF/IHC

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG2B Clone # MT 23.2

Format

BSA Free
View all MGMT Primary Antibodies »

Product Specifications

Immunogen

Recombinant human MGMT (O6-methylguanine-DNA methyltransferase) purified from E. coli [UniProt# P16455]

Reactivity Notes

This antibody is mouse reactivity negative.

Localization

Nuclear

Clonality

Monoclonal

Host

Mouse

Isotype

IgG2B

Theoretical MW

24 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Scientific Data Images for MGMT Antibody (MT 23.2) - BSA Free

Western Blot: MGMT Antibody (MT 23.2)BSA Free [NB100-168]

Western Blot: MGMT Antibody (MT 23.2)BSA Free [NB100-168]

Western Blot: MGMT Antibody (MT 23.2) [NB100-168] - Detection of MGMT in MCF7 lysate using NB100-168 at 1:1000 dilution.
Immunocytochemistry/ Immunofluorescence: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Immunocytochemistry/ Immunofluorescence: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Immunocytochemistry/Immunofluorescence: MGMT Antibody (MT 23.2) [NB100-168] - Detection of MGMT (Green) in Hela cells using NB100-168 at a 1:50 dilution. Nuclei (Blue) are counterstained using Hoechst 33258.
Immunohistochemistry-Paraffin: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Immunohistochemistry-Paraffin: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Immunohistochemistry-Paraffin: MGMT Antibody (MT 23.2) [NB100-168] - IHC analysis of a formalin fixed paraffin-embedded (FFPE) human kidney using 1:2000 conc. of MGMT antibody (clone MT 23.2) on a Bond Rx autostainer (Leica Biosystems). The assay involved 20 minutes of heat induced antigen retrieval (HIER) using 10mM sodium citrate buffer (pH 6.0) and endogenous peroxidase quenching with peroxide block. The sections were incubated with primary antibody for 30 minutes and Bond Polymer Refine Detection (Leica Biosystems) with DAB was used for signal development followed by counterstaining with hematoxylin. Whole slide scanning and capturing of representative images was performed using Aperio AT2 (Leica Biosystems). Nuclear staining of MGMT was observed. Staining was performed by Histowiz.
Flow Cytometry: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow Cytometry: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow Cytometry: MGMT Antibody (MT 23.2) [NB100-168] - An intracellular stain was performed on HeLa cells with MGMT Antibody (MT 23.2) antibody NB100-168 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 1 ug/mL for 30 minutes at room temperature, followed by mouse F(ab)2 IgG (H+L) PE-conjugated secondary antibody (F0102B, R&D Systems).
Immunocytochemistry/ Immunofluorescence: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Immunocytochemistry/ Immunofluorescence: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Immunocytochemistry/Immunofluorescence: MGMT Antibody (MT 23.2) [NB100-168] - MCF7 cells were fixed for 10 minutes using 10% formalin and then permeabilized for 5 minutes using 1X TBS + 0.5% Triton-X100. The cells were incubated with anti-MGMT (MT 23.2) NB100-168 at a 1:200 dilution overnight at 4C and detected with an anti-mouse Dylight 488 (Green) at a 1:500 dilution. Actin was counterstained with Phalloidin 568 (Red) at a 1:200 dilution. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 40X objective.
Flow (Intracellular): MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow (Intracellular): MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow (Intracellular): MGMT Antibody (MT 23.2) [NB100-168] - An intracellular stain was performed on Jurkat cells with MGMT Antibody (MT 23.2) antibody NB100-168 (blue) and a matched isotype control NBP2-27231 (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 1 ug/mL for 30 minutes at room temperature, followed by mouse F(ab)2 IgG (H+L) APC-conjugated secondary antibody (F0101B, R&D Systems).
Flow Cytometry: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow Cytometry: MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow Cytometry: MGMT Antibody (MT 23.2) [NB100-168] - Detection of MGMT in Human Jurkat Cell Line by Flow Cytometry. Human Jurkat cell line was stained with Mouse Anti- MGMT Monoclonal Antibody (Catalog # NB100-168, filled histogram), or Mouse IgG2B isotype control (Catalog # MAB0041, open histogram) followed by APC-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fixed with FlowX FoxP3 Fixation & Permeabilization Buffer Kit (Catalog # FC012).
Images may not be copied, printed or otherwise disseminated without express written permission of Novus Biologicals a bio-techne brand.
Flow (Intracellular): MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow (Intracellular): MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow (Intracellular): MGMT Antibody (MT 23.2) [NB100-168] - An intracellular stain was performed on Raji cells with MGMT Antibody (MT 23.2) antibody NB100-168AF647 (blue) and a matched isotype control NBP2-27231AF647 (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 2 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.
Flow (Intracellular): MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow (Intracellular): MGMT Antibody (MT 23.2) - BSA Free [NB100-168]

Flow (Intracellular): MGMT Antibody (MT 23.2) [NB100-168] - An intracellular stain was performed on Jurkat cells with MGMT Antibody (MT 23.2) antibody NB100-168AF647 (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeablized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.
Simple Western: MGMT Antibody (MT 23.2)BSA Free [NB100-168]

Simple Western: MGMT Antibody (MT 23.2)BSA Free [NB100-168]

Simple Western: MGMT Antibody (MT 23.2) [NB100-168] - Simple Western lane view shows a specific band for MGMT in 0.2 mg/ml of MCF-7 lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Applications for MGMT Antibody (MT 23.2) - BSA Free

Application
Recommended Usage

Flow Cytometry

1-2 ug/mL

Immunocytochemistry/ Immunofluorescence

1:500

Immunohistochemistry

1:500-1000

Immunohistochemistry-Paraffin

1:500-1:1000

Simple Western

1:1000

Western Blot

1:1000
Application Notes

This MGMT (MT 23.2) antibody is useful for Flow Cytometry, Immunocytochemistry/Immunofluorescence, Immunohistochemistry on paraffin-embedded sections and Western Blot. In WB, a band at approx. 24 kDa can be seen. In ICC/IF, strong staining has been seen in CEM cells and no signal has been seen in TK6 cells.

In Simple Western only 10 - 15 ul of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in MCF-7 lysate 0.2 mg/mL, separated by Size, antibody dilution of 1:1000, apparent MW was 26 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue.

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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: MGMT

MGMT (6-O-methylguanine-DNA methyltransferase) is a DNA repair enzyme with the ability to protect cells from carcinogenic effects of alkylating agents by removing adducts from the O(6) position of guanine. MGMT repairs alkylated guanine residues in DNA by stoichiometrically transferring the alkyl group at the O-6 position to a cysteine residue in the enzyme, and in this so called 'suicidal reaction', the enzyme is irreversibly inactivated. Localized in the nucleus, MGMT is ubiquitously expressed in normal human tissues with overexpression observed in several types of cancerous cells wherein it provides resistance against alkylating drugs. However, it is found epigenetically silenced in some cancers that include lymphoma, breast and prostate cancer, as well as retinoblastoma, wherein this silencing is thought to be due to promoter methylation which interferes with transcriptional factors binding. MGMT silencing has been linked to mutations in other tumor-related genes including silencing of p53 (non-small cell lung cancer and astrocytic tumors), k-ras gene mutations (gastric and colorectal cancer), and methylation of CDKN1A gene (encodes p21) as well as CDKN2A gene (encodes p16).

Long Name

O-6 Methylguanine-DNA Methyltransferase

Alternate Names

6-O-methylguanine-DNA methyltransferase, EC 2.1.1.63, methylated-DNA--protein-cysteine methyltransferase, methylguanine-DNA methyltransferase, O-6-methylguanine-DNA methyltransferase, O6-methylguanine-DNA methyltransferase, O-6-methylguanine-DNA-alkyltransferase

Entrez Gene IDs

4255 (Human); 17314 (Mouse)

Gene Symbol

MGMT

UniProt

P16455

Additional MGMT Products

Product Documents for MGMT Antibody (MT 23.2) - BSA Free

Certificate of Analysis

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Product Specific Notices for MGMT Antibody (MT 23.2) - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Related Research Areas

Cancer Biomarkers
Epigenetics

Citations for MGMT Antibody (MT 23.2) - BSA Free

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Protocols

View specific protocols for MGMT Antibody (MT 23.2) - BSA Free (NB100-168):




Western Blot Procedure

1. Run ~ 50 _g of whole cell extract on a PAGE gel (ie: 4-12% NuPAGE Bis-Tris gel).

2. Transfer the protein from the gel to a nitrocellulose membrane for 45-60 minutes.

3. Block the membrane with blocking buffer [1X TBS / 0.1% Tween-20 / 5% NFDM] for 1 hour at room temperature.

4. Rinse the membrane with 1X TBS, twice.

5. Dilute the 1_ antibody (cat# NB100-168, anti-MGMT) 1:1,000 to 1:3,000, in dilution buffer [1X TBS + 1% BSA].

6. Incubate the membrane with 1_ antibody for 1 hour at room temperature.

7. Wash the membrane with washing buffer [1X TBS + 0.05% Tween-20] 1 X 15 minutes, 3 x 5 minutes.

8. Dilute 2_ antibody [anti-mouse-HRP] in dilution buffer, add to the membrane, and incubate for 35 minutes at room temperature.

9. Wash the membrane with washing buffer 1 X 15 minutes, 3 x 5 minutes.

10. Develop the membrane with an ECL kit (ie: ChemiGlow Chemiluminescence reagent from AlphaInnotech].

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