Figure 1. Western blot analysis of MAGED4 using anti-MAGED4 antibody (MBS1759485).
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 Jurkat whole cell lysates,
Lane 2: human A549 whole cell lysates,
Lane 3: human HepG2 whole cell lysates,
Lane 4: human U2OS 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-MAGED4 antigen affinity purified polyclonal antibody (#MBS1759485) at 0.5ug/mL overnight at 4 degree C, 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 (#MBS176460) with Tanon 5200 system. A specific band was detected for MAGED4 at approximately 100 kDa. The expected band size for MAGED4 is at 100 kDa.
Figure 2. IHC analysis of MAGED4 using anti-MAGED4 antibody (MBS1759485).
MAGED4 was detected in a paraffin-embedded section of human adrenocortical adenoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2ug/ml rabbit anti-MAGED4 Antibody (MBS1759485) overnight at 4 degree C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (#MBS176453) with DAB as the chromogen.
Figure 3. IHC analysis of MAGED4 using anti-MAGED4 antibody (MBS1759485).
MAGED4 was detected in a paraffin-embedded section of human bladder urothelial carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2ug/ml rabbit anti-MAGED4 Antibody (MBS1759485) overnight at 4 degree C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37 degree C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (#MBS176453) with DAB as the chromogen.
Figure 4. IF analysis of MAGED4 using anti-MAGED4 antibody (MBS1759485).
MAGED4 was detected in an immunocytochemical section of A549 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (#MBS176582) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5ug/mL rabbit anti-MAGED4 Antibody (MBS1759485) overnight at 4 degree C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37 degree C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.
Figure 5. Flow Cytometry analysis of HepG2 cells using anti-MAGED4 antibody (MBS1759485).
Overlay histogram showing HepG2 cells stained with MBS1759485 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-MAGED4 Antibody (MBS1759485, 1ug/1x106 cells) for 30 min at 20 degree C. DyLight488 conjugated goat anti-rabbit IgG (MBS177405, 5-10ug/1x106 cells) was used as secondary antibody for 30 minutes at 20 degree C. Isotype control antibody (Green line) was rabbit IgG (1ug/1x106) used under the same conditions. Unlabelled sample (Red line) was also used as a control.
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