Anti-KAP1/TRIM28 Antibody Picoband® (monoclonal, 3H2)

Figure 1. Western blot analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
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 50ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates;
Lane 2: human PC-3 whole cell lysates;
Lane 3: human HEK293 whole cell lysates;
Lane 4: human A549 whole cell lysates;
Lane 5: human Jurkat whole cell lysates;
Lane 6: human THP-1 whole cell lysates;
Lane 7: rat PC-12 whole cell lysates;
Lane 8: mouse NIH/3T3 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-KAP1/TRIM28 antigen affinity purified monoclonal antibody (Catalog # M00409-1) 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-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 KAP1/TRIM28 at approximately 100KD. The expected band size for KAP1/TRIM28 is at 100KD.

Figure 2. IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 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-KAP1/TRIM28 Antibody (M00409-1) 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 KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 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-KAP1/TRIM28 Antibody (M00409-1) 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. IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 was detected in paraffin-embedded section of mouse small intestine 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-KAP1/TRIM28 Antibody (M00409-1) 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 5. IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 was detected in paraffin-embedded section of rat intestine 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-KAP1/TRIM28 Antibody (M00409-1) 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 6. IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 was detected in frozen section of human placenta tissue. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-KAP1/TRIM28 Antibody (M00409-1) 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 7. IF analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 was detected in paraffin-embedded section of human rectal carcinoma 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 2μg/mL mouse anti-KAP1/TRIM28 Antibody (M00409-1) 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. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Figure 8. IF analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 was detected in paraffin-embedded section of human breast 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 2μg/mL mouse anti-KAP1/TRIM28 Antibody (M00409-1) 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 9. IF analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody (M00409-1).
KAP1/TRIM28 was detected in immunocytochemical section of U20S 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 2μg/mL mouse anti-KAP1/TRIM28 Antibody (M00409-1) 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 10. Flow Cytometry analysis of A549 cells using anti- KAP1/TRIM28 antibody (M00409-1).
Overlay histogram showing A549 cells stained with M00409-1 (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 mouse anti- KAP1/TRIM28 Antibody (M00409-1, 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.