Anti-KAP1/TRIM28 Antibody (monoclonal, 9E3)

• Catalog Number: orb654268
• Category: Antibodies
• Description: Anti-KAP1/TRIM28 Antibody (monoclonal, 9E3). Tested in Flow Cytometry, IF, IHC, IHC-F, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.
• Clonality: Monoclonal
• Species/Host: Mouse
• Isotype: Mouse IgG2a
• Conjugation: Unconjugated
• Reactivity: Human, Mouse, Rat
• Form/Appearance: Lyophilized
• Concentration: Adding 0.2 ml of distilled water will yield a concentration of 500 μg/ml.
• Purification: Immunogen affinity purified.
• Immunogen: E.coli-derived human KAP1 recombinant protein (Position: A699-P835). Human KAP1 shares 94.9% amino acid (aa) sequence identity with both mouse and rat KAP1.
• UniProt ID: Q13263
• MW: 100 kDa
• Tested applications: FC, ICC, IF, IHC, IHC-Fr, WB
• Application notes: Western blot, 0.1-0.5μg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1μg/ml, Human, Mouse, Rat Immunohistochemistry (Frozen Section), 0.5-1μg/ml, Human, Rat Immunocytochemistry/Immunofluorescence, 2μg/ml, Human Immunofluorescence, 2μg/ml, Human Flow Cytometry (Fixed), 1-3μg/1x106 cells, Human. Add 0.2ml of distilled water will yield a concentration of 500ug/ml
• Cross Reactivity: No cross-reactivity with other proteins.
• Antibody Type: Primary Antibody
• Clone Number: 9E3
• Storage: Maintain refrigerated at 2-8°C for up to 2 weeks. For long term storage store at -20°C in small aliquots to prevent freeze-thaw cycles.
• Alternative names: E3 SUMO protein ligase TRIM28 antibody; E3 SUMO-pr
• Note: For research use only

Flow Cytometry analysis of A549 cells using anti-KAP1/TRIM28 antibody. Overlay histogram showing A549 cells (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 (1 µg/1x10^6 cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (5-10 µg/1x10^6 cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10^6) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.

IF analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody. KAP1/TRIM28 was detected in immunocytochemical section of U20S 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 mouse anti-KAP1/TRIM28 Antibody overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG 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.

IF analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody. 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 2 µg/mL mouse anti-KAP1/TRIM28 Antibody overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG 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.

IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody. 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 1 µg/ml mouse anti-KAP1/TRIM28 Antibody 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) with DAB as the chromogen.

IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody. KAP1/TRIM28 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 1 µg/ml mouse anti-KAP1/TRIM28 Antibody 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) with DAB as the chromogen.

IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody. 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 1 µg/ml mouse anti-KAP1/TRIM28 Antibody 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) with DAB as the chromogen.

IHC analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 antibody. 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 1 µg/ml mouse anti-KAP1/TRIM28 Antibody 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) with DAB as the chromogen.

Western blot analysis of KAP1/TRIM28 using anti-KAP1/TRIM28 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 50 ug 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 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 mouse anti-KAP1/TRIM28 antigen affinity purified monoclonal antibody 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 with Tanon 5200 system. A specific band was detected for KAP1/TRIM28 at approximately 100 KD. The expected band size for KAP1/TRIM28 is at 100 KD.