Anti-H2AFY2/MACROH2A2 Antibody Picoband™

Figure 1. Western blot analysis of H2AFY2/MACROH2A2 using anti-H2AFY2/MACROH2A2 antibody (A09931-1).
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 HepG2 whole cell lysates,
Lane 2: human SH-SY5Y whole cell lysates,
Lane 3: human PC-3 whole cell lysates,
Lane 4: human 293T whole cell lysates,
Lane 5: rat brain tissue lysates,
Lane 6: mouse brain tissue 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-H2AFY2/MACROH2A2 antigen affinity purified polyclonal antibody (Catalog # A09931-1) at 0.5 μg/mL overnight at 4°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 (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for H2AFY2/MACROH2A2 at approximately 40 kDa. The expected band size for H2AFY2/MACROH2A2 is at 40 kDa.

Figure 2. IHC analysis of H2AFY2/MACROH2A2 using anti-H2AFY2/MACROH2A2 antibody (A09931-1).
H2AFY2/MACROH2A2 was detected in a paraffin-embedded section of human liver cancer 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 2 μg/ml rabbit anti-H2AFY2/MACROH2A2 Antibody (A09931-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 3. IHC analysis of H2AFY2/MACROH2A2 using anti-H2AFY2/MACROH2A2 antibody (A09931-1).
H2AFY2/MACROH2A2 was detected in a paraffin-embedded section of human tonsil 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 2 μg/ml rabbit anti-H2AFY2/MACROH2A2 Antibody (A09931-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 4. IHC analysis of H2AFY2/MACROH2A2 using anti-H2AFY2/MACROH2A2 antibody (A09931-1).
H2AFY2/MACROH2A2 was detected in a paraffin-embedded section of human 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 2 μg/ml rabbit anti-H2AFY2/MACROH2A2 Antibody (A09931-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

Figure 5. IF analysis of H2AFY2/MACROH2A2 using anti-H2AFY2/MACROH2A2 antibody (A09931-1) and anti-Beta Tubulin antibody (M01857-3).
H2AFY2/MACROH2A2 was detected in immunocytochemical section of U2OS cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-H2AFY2/MACROH2A2 Antibody (A09931-1) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) and DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Figure 6. Flow Cytometry analysis of PC-3 cells using anti-H2AFY2/MACROH2A2 antibody (A09931-1).
Overlay histogram showing PC-3 cells stained with A09931-1 (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-H2AFY2/MACROH2A2 Antibody (A09931-1, 1 μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10 μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1 μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.