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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 1 µg/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 1 µg/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 1 µg/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 1 µg/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 1 µg/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 2 µg/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 2 µg/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 2 µg/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).The cells were blocked with 10% normal goat serum. And then incubated with mouse anti-KAP1/TRIM28 Antibody (M00409-1, 1 µg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-mouse IgG (BA1126, 5-10 µg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was mouse IgG (1 µg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

SPECIFICATION
CITATIONS
PROTOCOLS
BACKGROUND

Product Information
Application Applications Legend: WB=Western Blot IHC=Immunohistochemistry IHC-P=Immunohistochemistry (Paraffin-embedded Sections) IHC-F=Immunohistochemistry (Frozen Sections) IF=Immunofluorescence FC=Flow Cytopmetry IC=Immunochemistry ICC=Immunocytochemistry E=ELISA IP=Immunoprecipitation DB=Dot Blot CHIP=Chromatin Immunoprecipitation FA=Fluorescence Assay IEM=Immuno electron microscopy EIA=Enzyme Immunoassay	WB, IHC, IHC-F, IF, ICC, FC
Primary Accession	Q13263
Host	Mouse
Isotype	Mouse IgG2b
Reactivity	Rat, Human, Mouse
Clonality	Monoclonal
Format	Lyophilized
Description	Anti-KAP1/TRIM28 Antibody Picoband™ (monoclonal, 3H2) . Tested in Flow Cytometry, IF, IHC, IHC-F, ICC, WB applications. This antibody reacts with Human, Mouse, Rat.
Reconstitution	Add 0.2ml of distilled water will yield a concentration of 500ug/ml.

Additional Information
Gene ID	10155
Other Names	Transcription intermediary factor 1-beta, TIF1-beta, E3 SUMO-protein ligase TRIM28, 2.3.2.27, KRAB-associated protein 1, KAP-1, KRAB-interacting protein 1, KRIP-1, Nuclear corepressor KAP-1, RING finger protein 96, RING-type E3 ubiquitin transferase TIF1-beta, Tripartite motif-containing protein 28, TRIM28 (HGNC:16384), KAP1, RNF96, TIF1B
Calculated MW	100 kDa
Application Details	Western blot, 0.1-0.5 µg/ml, Human, Mouse, Rat Immunohistochemistry (Paraffin-embedded Section), 0.5-1 µg/ml, Human, Mouse, Rat Immunohistochemistry (Frozen Section), 0.5-1 µg/ml, Human, Rat Immunocytochemistry/Immunofluorescence, 2 µg/ml, Human Immunofluorescence, 2 µg/ml, Human Flow Cytometry, 1-3 µg/1x10^6 cells, Human
Subcellular Localization	Nucleus.
Tissue Specificity	Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.
Contents	Each vial contains 4mg Trehalose, 0.9mg NaCl, 0.2mg Na₂HPO₄, 0.05mg NaN₃.
Clone Names	Clone: 3H2
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.
Purification	Immunogen affinity purified.
Cross Reactivity	No cross-reactivity with other proteins.
Storage	Store at -20˚C for one year from date of receipt. After reconstitution, at 4˚C for one month. It can also be aliquotted and stored frozen at -20˚C for six months. Avoid repeated freeze-thaw cycles.

Protein Information
Name	TRIM28 (HGNC:16384)
Synonyms	KAP1, RNF96, TIF1B
Function	Nuclear corepressor for KRAB domain-containing zinc finger proteins (KRAB-ZFPs). Mediates gene silencing by recruiting CHD3, a subunit of the nucleosome remodeling and deacetylation (NuRD) complex, and SETDB1 (which specifically methylates histone H3 at 'Lys-9' (H3K9me)) to the promoter regions of KRAB target genes. Enhances transcriptional repression by coordinating the increase in H3K9me, the decrease in histone H3 'Lys-9 and 'Lys-14' acetylation (H3K9ac and H3K14ac, respectively) and the disposition of HP1 proteins to silence gene expression. Recruitment of SETDB1 induces heterochromatinization. May play a role as a coactivator for CEBPB and NR3C1 in the transcriptional activation of ORM1. Also a corepressor for ERBB4. Inhibits E2F1 activity by stimulating E2F1-HDAC1 complex formation and inhibiting E2F1 acetylation. May serve as a partial backup to prevent E2F1-mediated apoptosis in the absence of RB1. Important regulator of CDKN1A/p21(CIP1). Has E3 SUMO-protein ligase activity toward itself via its PHD-type zinc finger. Also specifically sumoylates IRF7, thereby inhibiting its transactivation activity. Ubiquitinates p53/TP53 leading to its proteasomal degradation; the function is enhanced by MAGEC2 and MAGEA2, and possibly MAGEA3 and MAGEA6. Mediates the nuclear localization of KOX1, ZNF268 and ZNF300 transcription factors. In association with isoform 2 of ZFP90, is required for the transcriptional repressor activity of FOXP3 and the suppressive function of regulatory T-cells (Treg) (PubMed:23543754). Probably forms a corepressor complex required for activated KRAS-mediated promoter hypermethylation and transcriptional silencing of tumor suppressor genes (TSGs) or other tumor-related genes in colorectal cancer (CRC) cells (PubMed:24623306). Required to maintain a transcriptionally repressive state of genes in undifferentiated embryonic stem cells (ESCs) (PubMed:24623306). In ESCs, in collaboration with SETDB1, is also required for H3K9me3 and silencing of endogenous and introduced retroviruses in a DNA-methylation independent-pathway (By similarity). Associates at promoter regions of tumor suppressor genes (TSGs) leading to their gene silencing (PubMed:24623306). The SETDB1-TRIM28-ZNF274 complex may play a role in recruiting ATRX to the 3'-exons of zinc- finger coding genes with atypical chromatin signatures to establish or maintain/protect H3K9me3 at these transcriptionally active regions (PubMed:27029610).
Cellular Location	Nucleus Note=Associated with centromeric heterochromatin during cell differentiation through CBX1 (By similarity). Localizes to sites of DNA damage (PubMed:25593309). {ECO:0000250\|UniProtKB:Q62318, ECO:0000269\|PubMed:25593309}
Tissue Location	Expressed in all tissues tested including spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocytes.

Research Areas

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Application Protocols

Provided below are standard protocols that you may find useful for product applications.

Western Blot Protocol

Blocking Peptides Protocol

Dot Blot Protocol

Immunohistochemistry Protocol

Immunofluorescence Protocol

Immunoprecipitation Protocol

Flow Cytomety Protocol

Cell Culture Protocol

Background

Tripartite motif-containing 28 (TRIM28), also known as transcriptional intermediary factor 1β (TIF1β) and KAP1 (KRAB-associated protein-1), is a protein that in humans is encoded by the TRIM28 gene. The protein encoded by this gene mediates transcriptional control by interaction with the Kruppel-associated box repression domain found in many transcription factors. The protein localizes to the nucleus and is thought to associate with specific chromatin regions. KAP1 is a ubiquitously expressed protein involved in many critical functions including: transcriptional regulation, cellular differentiation and proliferation, DNA damage repair, viral suppression, and apoptosis. Its functionality is dependent upon post-translational modifications. Phosphorylation of KAP1 acts as a deactivator of the protein in many of its mechanisms while sumoylation acts as an activator.

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Cat# ABO14933

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