Overview

  • Product name
    Anti-MGMT antibody [MT3.1]
    See all MGMT primary antibodies
  • Description
    Mouse monoclonal [MT3.1] to MGMT
  • Host species
    Mouse
  • Tested applications
    Suitable for: IHC-Fr, WB, IHC-FoFr, ICC, Flow Cyt, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant full length protein corresponding to Human MGMT.

  • Positive control
    • Tonsil

Properties

Applications

Our Abpromise guarantee covers the use of ab39253 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-Fr 1/50.
WB 1/500. Detects a band of approximately 21 kDa.
IHC-FoFr 1/25 - 1/50.
ICC Use at an assay dependent concentration.
Flow Cyt 1/50.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

ICC/IF Use at an assay dependent concentration.
IHC-P 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

  • 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
  • Database links
  • Alternative names
    • 6 O methylguanine DNA methyltransferase antibody
    • 6-O-methylguanine-DNA methyltransferase antibody
    • Agat antibody
    • AGT antibody
    • AI267024 antibody
    • EC 2.1.1.63 antibody
    • Methylated DNA protein cysteine methyltransferase antibody
    • Methylated-DNA--protein-cysteine methyltransferase antibody
    • Methylguanine DNA methyltransferase antibody
    • MGC107020 antibody
    • MGMT antibody
    • MGMT_HUMAN antibody
    • O 6 methylguanine DNA alkyltransferase antibody
    • O 6 methylguanine DNA methyltransferase antibody
    • O-6-methylguanine-DNA methyltransferase antibody
    • O-6-methylguanine-DNA-alkyltransferase antibody
    see all

Images

  • 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 - ab39253 observed at 22 kDa. Red - loading control, ab181602, observed at 37 kDa.
    ab39253 was shown to specifically react with MGMT when MGMT knockout samples were used. Wild-type and MGMT knockout samples were subjected to SDS-PAGE. ab39253 and ab181602 (loading control to GAPDH) were diluted 1/500 and 1/10 000 and incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

  • ab39253 staining MGMT in Human stomach tissue sections by IHC-Fr (frozen sections). Tissue was fixed with paraformaldehyde and blocked with 5% serum for 1 hour at 23°C. Samples were incubated with primary antibody (1/50 in blocking buffer) for 1 hour at 23°C. An undiluted HRP-conjugated goat anti-mouse IgG polyclonal was used as secondary antibody.

    See Abreview

  • ab39253 staining MGMT in human stomach tissue sections by Immunohistochemistry. Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed using 10mM citrate buffer pH6.0. Samples were then blocked with 5% serum for 1 hour at 23°C followed by incubation with the primary antibody, at a 1/100 dilution, for 1 hour at 23°C. An undiluted HRP-conjugated goat anti-mouse IgG polyclonal was used as secondary antibody.

    See Abreview

  • Overlay histogram showing Jurkat cells stained with ab39253 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab39253, 1/50 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • All lanes : Anti-MGMT antibody [MT3.1] (ab39253) at 1/500 dilution (for 16 hours at 4°C)

    All lanes : Human OE33 cells - whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Rabbit Anti-Mouse IgG H&L (HRP) (ab6728) at 1/1000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Observed band size: 21 kDa
    why is the actual band size different from the predicted?


    Exposure time: 30 seconds


    Blocking Step: 5% BSA for 1 hour at 23°C

    See Abreview

  • Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human tonsil tissue, staining MGMT with ab39253.

References

This product has been referenced in:
  • Zink LM  et al. H3.Y discriminates between HIRA and DAXX chaperone complexes and reveals unexpected insights into human DAXX-H3.3-H4 binding and deposition requirements. Nucleic Acids Res 45:5691-5706 (2017). Read more (PubMed: 28334823) »
  • Wang K  et al. Hedgehog/Gli1 signaling pathway regulates MGMT expression and chemoresistance to temozolomide in human glioblastoma. Cancer Cell Int 17:117 (2017). Read more (PubMed: 29225516) »
See all 9 Publications for this product

Customer reviews and Q&As

1-10 of 11 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HeLa)
Permeabilization
Yes - 0.5% Triton X-100
Specification
HeLa
Fixative
Paraformaldehyde

Dr. Kirk Mcmanus

Verified customer

Submitted Jul 03 2018

Answer

Thank you for contacting Abcam.

We do not have expiration dates for our products, but we do guarantee our products for 6 months if stored according to the datasheet. Although we cannot guarantee it, based on our in-house testing if the antibody is stored at 4C, then it should be good for a year.

If there is anything else I can help you with, please let me know.

Free RabMab with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Read More

Answer

Thank you very much for your reply. My colleague is out of the office today but I'll be happy to help you with this.

We don't currently have blocking peptides available for ab105340 or ab56540, but I'm in the process of adding the peptide for ab105340 to our catalog for you. Since ab56540 was raised against the full length protein instead of a short peptide, there is no blocking peptide available for this antibody.

Blocking peptides are often used in Western blot to verify specificity of a band or bands. The amino acid sequence of the blocking peptide is the same as that of the immunogenic peptide. By pre-treating the antibody with an excess of the blocking peptide, the specific binding sites of the antibody should be "blocked", so that if the antibody-peptide complex is used in the Western blot instead of the pure antibody, the specific bands will not appear on the blot. Here is the blocking protocol, just for further clarification of the concept-

https://www.abcam.com/index.html?pageconfig=resource&rid=11378

You couldin theoryuse a full-length protein to block ab56540, but this requires quite a lot of protein and may not be successful or give conclusive results. Knock-out testing or mass spectrometry of the suspected band may be a more efficient and definitive control.

I hope that this will be helpful. We will be back in touch as soon as we have the blocking peptide of ab105340 available for you.

Read More

Answer

Thank you for your reply. The publications that are known to reference an Abcam product can be found on our online datasheets on the specific references tab. Below are PubMed IDs for the antibodies you are interested in.

MGMT (ab39253) - 21752281, 17508213

ABH2 (ab105340) - no known references

APNG (ab55461) - 22496614, 20064251

ABH3 (ab56540) - no known references

I hope this helps, please let me know if you need any additional information or assistance.

Read More

Answer

Thank you for contacting us.

The final dilutions should be optimized for your particular samples and protocols, but the following would be my recommended starting dilutions for WB with an overnight incubation:

MGMT (ab39253) - 1:500

ABH2 (ab105340) - 1:1000

APNG (ab55461) - 1:500

ABH3 (ab56540) - 1:500

I hope this helps, please let me know if you need any additional information or assistance.

Read More

Question
Answer

Thank you for contacting us and sorry for the delay in getting back to you.


I can confirm that theaverage concentration of this antibody is 66.6 ug/ml.


I hope this information is of help. If you require any further information please do not hesitate to contact us again.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (OE33 cells)
Loading amount
20 µg
Specification
OE33 cells
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Aug 27 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (AGS Gastric Carcinoma cells)
Specification
AGS Gastric Carcinoma cells
Fixative
Formaldehyde
Permeabilization
Yes - 0.025% Triton-X in TBS
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jun 02 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Human Cultured Cells (AGS Gastric Carcinoma cells)
Specification
AGS Gastric Carcinoma cells
Fixative
Formaldehyde
Permeabilization
Yes - 0.025% Triton-X in TBS
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted May 19 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Stomach)
Specification
Stomach
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: 10mM citrate pH6.0
Permeabilization
No
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Mar 24 2010

1-10 of 11 Abreviews or Q&A

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