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MGMT Recombinant Rabbit Monoclonal Antibody [JJ089-6]

Usd: 385

Specification

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Catalog# ET1701-55

MGMT Recombinant Rabbit Monoclonal Antibody [JJ089-6]

Application

  • WB

  • IHC-P

  • IF-Cell

  • FC(Intra)

Reactivity

  • Human

BSA and Azide free
Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

MGMT Recombinant Rabbit Monoclonal Antibody [JJ089-6]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Recombinant protein within Human MGMT aa 1-140 / 207.

Species Reactivity

Human

Validated Applications

WB, IHC-P, IF-Cell, FC(Intra)

Target Molecular Weight

Predicted band size: 22 kDa

Positive Control

MCF7 cell lysate, Jurkat cell lysate, HCT 116 cell lysate, HeLa cell lysate, MCF7, human tonsil tissue.

Conjugation

unconjugated

Clone Number

JJ089-6

RRID

AB_3070220

Product Features

Form

Liquid

Concentration

1 mg/mL.(The concentration of this product may be batch-dependent)

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:2,000-1:5,000

  • IHC-P

  • 1:50-1:200

  • IF-Cell

  • 1:100

  • FC(Intra)

  • 1:1,000

Target

Function

MGMT (O6-methylguanine-DNA methyltransferase) is transcriptionally activated in response to DNA damage and functions to repair mutagenic and cytotoxic O6-alkylguanine lesions caused by carcinogens or cytostatic drugs. MGMT induction by ionising radiation does not occur in p53-deficient mice, suggesting that MGMT induction may require p53. Similarly, MGMT mRNA and protein were shown to be inducible by ionising radiation, only in cell lines that express functional p53, and not in cell lines that do not express wild type p53. In contrast, high MGMT activity was associated with the presence of mutant p53, in a study of oral cancer cell lines. Similarly, MGMT activity was significantly lower in ovarian tumors with wildtype p53 than in tumors with mutant p53, supporting the view that wildtype p53 down-regulates the basal MGMT promoter.

Background References

1. Viel T et al. Optimizing Glioblastoma Temozolomide Chemotherapy Employing Lentiviral-based Anti-MGMT shRNA Technology. Mol Ther 21:570-9 (2013).

2. Viel T et al. Early assessment of the efficacy of temozolomide chemotherapy in experimental glioblastoma using [18F]FLT-PET imaging. PLoS One 8:e67911 (2013).

Sequence Similarity

Belongs to the MGMT family.

Subcellular Location

Nucleus.

UNIPROT

SwissProt: P16455 Human

Synonyms

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

Expand

Images

  • <span style="font-weight: bold;">☑ Relative expression (RE)</span><br /><br />Western blot analysis of MGMT on different lysates with Rabbit anti-MGMT antibody (<a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>) at 1/5,000 dilution.<br /><br />Lane 1: MCF7 (Human breast cancer cell) cell lysate<br />Lane 2: MDA-MB-231 (Human breast cancer cell) cell lysate<br />Lane 3: Jurkat (Human T-lymphoblastic cells) cell lysate<br />Lane 4: HCT 116 (Human colon cancer cell) cell lysate<br />Lane 5: MOLT-4 (Human acute T-Cell leukemia cell) cell lysate<br />Lane 6: HeLa (Human cervical adenocarcinoma cell) cell lysate<br />Lane 7: HT-29 (Human colorectal adenocarcinoma cell) cell lysate<br /><br />Lysates/proteins at 15 µg/Lane.<br />Exposure time: 25 seconds; ECL: K1801<br /><br />MDA-MB-231 exhibits low MGMT expression (PMID: 26420420).<br /><br />Blocking: 5% NFDM/TBST, 1 hour at room temperature<br />Primary antibody: <a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>, 1/5,000 in primary antibody dilution buffer (<a href="/products/K1803" style="font-weight: bold;text-decoration: underline;">K1803</a>), overnight at 4 ℃<br />Secondary antibody: Goat anti-Rabbit IgG-HRP (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature<br /><br />Predicted band size: 22 kDa<br />Observed band size: 22 kDa

    ☑ Relative expression (RE)

    Western blot analysis of MGMT on different lysates with Rabbit anti-MGMT antibody (ET1701-55) at 1/5,000 dilution.

    Lane 1: MCF7 (Human breast cancer cell) cell lysate
    Lane 2: MDA-MB-231 (Human breast cancer cell) cell lysate
    Lane 3: Jurkat (Human T-lymphoblastic cells) cell lysate
    Lane 4: HCT 116 (Human colon cancer cell) cell lysate
    Lane 5: MOLT-4 (Human acute T-Cell leukemia cell) cell lysate
    Lane 6: HeLa (Human cervical adenocarcinoma cell) cell lysate
    Lane 7: HT-29 (Human colorectal adenocarcinoma cell) cell lysate

    Lysates/proteins at 15 µg/Lane.
    Exposure time: 25 seconds; ECL: K1801

    MDA-MB-231 exhibits low MGMT expression (PMID: 26420420).

    Blocking: 5% NFDM/TBST, 1 hour at room temperature
    Primary antibody: ET1701-55, 1/5,000 in primary antibody dilution buffer (K1803), overnight at 4 ℃
    Secondary antibody: Goat anti-Rabbit IgG-HRP (HA1001), 1/50,000 in 5% NFDM/TBST, 1 hour at room temperature

    Predicted band size: 22 kDa
    Observed band size: 22 kDa

  • <span style="font-weight: bold;">☑ Knockdown (KD)</span><br /><br />Western blot analysis of MGMT on different lysates with Rabbit anti-MGMT antibody (<a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>) at 1/2,000 dilution.<br /><br />Lane 1: HAP1-parental cell lysate<br />Lane 2: HAP1-MGMT KD cell lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 22 kDa<br />Observed band size: 22 kDa<br /><br />Exposure time: 2 minutes 40 seconds; ECL: K1801;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>) at 1/2,000 dilution was used in primary antibody dilution (<a href="/products/K1803" style="font-weight: bold;text-decoration: underline;">K1803</a>) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Knockdown (KD)

    Western blot analysis of MGMT on different lysates with Rabbit anti-MGMT antibody (ET1701-55) at 1/2,000 dilution.

    Lane 1: HAP1-parental cell lysate
    Lane 2: HAP1-MGMT KD cell lysate

    Lysates/proteins at 10 µg/Lane.

    Predicted band size: 22 kDa
    Observed band size: 22 kDa

    Exposure time: 2 minutes 40 seconds; ECL: K1801;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1701-55) at 1/2,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of MCF7 cells labeling MGMT with Rabbit anti-MGMT antibody (<a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-MGMT antibody (<a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/HA601187" style="font-weight: bold;text-decoration: underline;">HA601187</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of MCF7 cells labeling MGMT with Rabbit anti-MGMT antibody (ET1701-55) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-MGMT antibody (ET1701-55) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

  • Application: Flow Cytometry (Intra)<br /><br />Species: Human<br />Sample: MCF7 (Human breast cancer cell)<br /><br />Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.<br />Permeabilization: 0.1% Tween-20, 15 minutes at room temperature.<br />Blocking: 1% BSA + 10% normal goat serum, 15 minutes at room temperature.<br />Antibody dilution buffer: 1x PBS.<br /><br />Primary antibody: <a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>(1 μg/mL, Red) compared with Rabbit IgG Isotype Control (<a href="/products/HA722127" style="font-weight: bold;text-decoration: underline;">HA722127</a>, Green), 15 minutes at room temperature.<br />Secondary antibody: Goat Anti-Rabbit IgG (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>), 15 minutes at room temperature.

    Application: Flow Cytometry (Intra)

    Species: Human
    Sample: MCF7 (Human breast cancer cell)

    Fixation: 4% Paraformaldehyde, 15 minutes at room temperature.
    Permeabilization: 0.1% Tween-20, 15 minutes at room temperature.
    Blocking: 1% BSA + 10% normal goat serum, 15 minutes at room temperature.
    Antibody dilution buffer: 1x PBS.

    Primary antibody: ET1701-55(1 μg/mL, Red) compared with Rabbit IgG Isotype Control (HA722127, Green), 15 minutes at room temperature.
    Secondary antibody: Goat Anti-Rabbit IgG (iFluor™ 488, HA1121), 15 minutes at room temperature.

  • Application: Immunohistochemistry (IHC-P)<br /><br />Species: Human<br />Tissue: Tonsil<br />Sample: Paraffin-embedded section<br /><br />Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.<br /><br />Wash buffer: 1× TBST<br />Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.<br />Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.<br />Primary antibody: <a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>, 1/200, 1 hour at room temperature.<br />Secondary antibody: <a href="/products/HA1119" style="font-weight: bold;text-decoration: underline;">HA1119</a>, 20 minutes at room temperature.

    Application: Immunohistochemistry (IHC-P)

    Species: Human
    Tissue: Tonsil
    Sample: Paraffin-embedded section

    Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

    Wash buffer: 1× TBST
    Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
    Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
    Primary antibody: ET1701-55, 1/200, 1 hour at room temperature.
    Secondary antibody: HA1119, 20 minutes at room temperature.

  • Application: Immunohistochemistry (IHC-P)<br /><br />Species: Human<br />Tissue: Lung carcinoma<br />Sample: Paraffin-embedded section<br /><br />Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.<br /><br />Wash buffer: 1× TBST<br />Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.<br />Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.<br />Primary antibody: <a href="/products/ET1701-55" style="font-weight: bold;text-decoration: underline;">ET1701-55</a>, 1/50, 1 hour at room temperature.<br />Secondary antibody: <a href="/products/HA1119" style="font-weight: bold;text-decoration: underline;">HA1119</a>, 20 minutes at room temperature.

    Application: Immunohistochemistry (IHC-P)

    Species: Human
    Tissue: Lung carcinoma
    Sample: Paraffin-embedded section

    Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

    Wash buffer: 1× TBST
    Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
    Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
    Primary antibody: ET1701-55, 1/50, 1 hour at room temperature.
    Secondary antibody: HA1119, 20 minutes at room temperature.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation