Anti-TIF1 gamma/TRIM33 Antibody Picoband™ (monoclonal, 8I8)

Figure 1. Western blot analysis of TIF1 gamma/TRIM33 using anti-TIF1 gamma/TRIM33 antibody (M03133-2).
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 50ug of sample under reducing conditions.
Lane 1: human MCF-7 whole cell lysates,
Lane 2: human SW620 whole cell lysates,
Lane 3: human K562 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-TIF1 gamma/TRIM33 antigen affinity purified monoclonal antibody (Catalog # M03133-2) 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-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for TIF1 gamma/TRIM33 at approximately 150KD. The expected band size for TIF1 gamma/TRIM33 is at 150KD.

Figure 2. IHC analysis of TIF1 gamma/TRIM33 using anti TIF1 gamma/TRIM33 antibody (M03133-2).
TIF1 gamma/TRIM33 was detected in paraffin-embedded section of human mammary 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 1μg/ml mouse anti-TIF1 gamma/TRIM33 Antibody (M03133-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

Figure 3. IHC analysis of TIF1 gamma/TRIM33 using anti TIF1 gamma/TRIM33 antibody (M03133-2).
TIF1 gamma/TRIM33 was detected in paraffin-embedded section of human rectal 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 1μg/ml mouse anti-TIF1 gamma/TRIM33 Antibody (M03133-2) overnight at 4°C. Biotinylated goat anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1021) with DAB as the chromogen.

Figure 4. IF analysis of TIF1 gamma/TRIM33 using anti-TIF1 gamma/TRIM33 antibody (M03133-2).
TIF1 gamma/TRIM33 was detected in immunocytochemical section of Hela cells. 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 4μg/mL mouse anti-TIF1 gamma/TRIM33 Antibody (M03133-2) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°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 HL-60 cells using anti- TIF1 gamma/TRIM33 antibody (M03133-2).
Overlay histogram showing HL-60 cells stained with M03133-2 (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 mouse anti-TIF1 gamma/TRIM33 Antibody (M03133-2, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.