E.coli-derived human MGMT recombinant protein (Position: D2-N207). Human MGMT shares 70% and 69% amino acid (aa) sequence identity with mouse and rat MGMT, respectively.
Each vial contains antibody formulated with stabilizing components, 0.9mg NaCl, 0.2mg Na2HPO4, 0.01mg NaN3. *This antibody is supplied in a stabilized formulation. Compatibility with conjugation reactions depends on the chemistry of the conjugation method
Western blot, 0.1-0.5µg/ml, Human Immunohistochemistry (Paraffin-embedded Section), 2-5µg/ml, Human Immunocytochemistry, 0.5-1µg/ml, Human Immunocytochemistry/Immunofluorescence, 2µg/ml, Human Flow Cytometry (Fixed), 1-3µg/1x10 6 cells, Human
Flow Cytometry analysis of THP-1 cells using anti-MGMT antibody. Overlay histogram showing THP-1 cells (Blue line). The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-MGMT Antibody (1 µg/1x10 6 cells) for 30 min at 20C. DyLight488 conjugated goat anti-rabbit IgG (5-10 µg/1x10 6 cells) was used as secondary antibody for 30 minutes at 20C. Isotype control antibody (Green line) was rabbit IgG (1 µg/1x10 6) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
Flow Cytometry analysis of THP-1 cells using anti-MGMT antibody (Blue line
ICC analysis of MGMT using anti-MGMT antibody. MGMT was detected in immunocytochemical section of human MCF-7 cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 1 µg/ml rabbit anti-MGMT Antibody overnight at 4C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37C. The section was developed using Strepavidin-Biotin-Complex (SABC) with DAB as the chromogen.
IF analysis of MGMT using anti-MGMT antibody. MGMT was detected in immunocytochemical section of MCF-7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 µg/mL rabbit anti-MGMT Antibody overnight at 4C. DyLight488 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IF analysis of MGMT using anti-MGMT antibody. MGMT was detected in immunocytochemical section of MCF7 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2 µg/mL rabbit anti-MGMT Antibody overnight at 4C. DyLight594 Conjugated Goat Anti-Rabbit IgG was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
IHC analysis of MGMT using anti-MGMT antibody. MGMT was detected in a paraffin-embedded section of human colorectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 µg/ml rabbit anti-MGMT Antibody overnight at 4C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit with DAB as the chromogen.
Western blot analysis of MGMT using anti-MGMT antibody. Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions. Lane 1: human CACO-2 whole cell lysates, Lane 2: human Raji whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-MGMT antigen affinity purified polyclonal antibody at 0.5 µg/mL overnight at 4C, then washed with TBS-0.1% Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit with Tanon 5200 system. A specific band was detected for MGMT at approximately 22 kDa. The expected band size for MGMT is at 22 kDa.
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