Anti-KAP1 抗体 [20C1]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [20C1] (AB22553)

IHC image of KAP1 staining in Human normal spleen formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab22553, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• ICC

Supplier Data

Immunocytochemistry - Anti-KAP1 antibody [20C1] (AB22553)

Immunofluorescence analysis of TIF1 beta was performed using 70% confluent log phase HeLa (Human cervix adenocarcinoma epithelial cell) cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab22553 at 2 μg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1 : 2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade^® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Rhodamine Phalloidin. Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

• WB

Lab

Western blot - Anti-KAP1 antibody [20C1] (AB22553)

Western blot : Anti-KAP1 antibody [20C1] ab22553 staining at 1/500 dilution, shown in green; Rabbit anti GAPDH ab181602 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 110 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in TRIM28 knockout HCT 116 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Mouse 800CW and Goat anti-Rabbit 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-KAP1 antibody [20C1] (ab22553) at 1/500 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

Western blot - Human TRIM28 knockout HCT116 cell line (ab289073) at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

A-431 at 20 µg

Secondary

All lanes:

Goat anti-Mouse 800CW & Goat anti-Rabbit 680RD at 1/20000 dilution

Predicted band size: 89 kDa

Observed band size: 110 kDa,36 kDa

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• WB

Supplier Data

Western blot - Anti-KAP1 antibody [20C1] (AB22553)

Western blot analysis of TRIM28 was performed by loading 10 g of whole cell human BJ fibroblast protein lysate and run on a 4-12% BTE gel. Proteins were transferred to PVDF membrane. Membrane was blocked in 5% non-fat milk in TBST. TRIM28 was detected at approximately 90 kDa using ab22553 at a dilution of 1 : 500 (2 μg/mL) in 5% milk, followed by a 1 : 10,000 dilution of anti-mouse HRP. Chemiluminescent detection was performed using SuperSignal West Pico PLUS substrate.

All lanes:

Western blot - Anti-KAP1 antibody [20C1] (ab22553) at 1/500 dilution

All lanes:

Whole cell human BJ fibroblast protein lysate at 10 µg

Predicted band size: 88 kDa

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• WB

CiteAb

Western blot - Anti-KAP1 antibody [20C1] (AB22553)

Western Blotting using Anti-KAP1 antibody [20C1], ab22553. Publication image from Li, W. et al., 2019, Nat Commun, 31350386. Legend direct from paper.

DGCR8 forms a complex with heterochromatin proteins and Lamin B1. a Co-immunoprecipitation analysis of KAP1, Lamin B1, HP1α, and HP1γ with exogenous Flag-tagged DGCR8 protein in HEK293T cells. b Co-immunoprecipitation analysis of KAP1, Lamin B1, HP1α, and HP1γ with endogenous DGCR8 protein in WT hMSCs. The HP1α and HP1γ bands pulled down by IP with an anti-DGCR8 antibody were indicated by asterisk. c Enrichment of DGCR8 within the region ofα-Sat or Sat2 measured by ChIP-qPCR. Data were presented as mean ± SEM. n = 3 wells per condition, **p < 0.01, ***p < 0.001. d Left, enrichment of H3K9me3 ChIP-seq signals ranging from 21 kb upstream to 21 kb downstream of heterochromatin (HC) regions in WT and DR8dex2 hMSCs. Right, heatmap showing the enrichment of H3K9me3 signals around HC regions (from 21 kb upstream to 21 kb downstream) in WT and DR8dex2 hMSCs. e Left, enrichment of H3K9me3 ChIP-seq signals ranging from 21 kb upstream to 21 kb downstream of lamina-associated domain (LAD) regions in WT and DR8dex2 hMSCs. Right, heatmap showing the enrichment of H3K9me3 signals around LAD regions (from 21 kb upstream to 21 kb downstream) in WT and DR8dex2 hMSCs. f Enrichment of H3K9me3 within the region ofα-Sat or Sat2 in WT and DR8dex2 hMSCs measured by ChIP-qPCR. Data were presented as mean ± SEM. n = 3 wells per condition, ***p < 0.001. g Quantitative RT-PCR analysis of centromeric element transcripts in WT and DR8dex2 hMSCs. Data were presented as mean ± SEM. n = 3 wells per condition, *p < 0.05, **p < 0.01, ***p < 0.001. h Sketch map of “H3K9me3 mountain” distribution over 23 chromosomes. Blue lines indicate 106 “H3K9me3 mountains” present in WT hMSCs. Red arrowheads indicate 62 “H3K9me3 mountains” lost in DR8dex2 hMSCs. Blue circles indicate the centromeres of chromosomes. i Enrichment of H3K9me3 ChIP-seq signals ranging from 21 kb upstream to 21 kb downstream of heterochromatin (HC) regions in DR8dex2 hMSCs transduced with Luc, DR8-WT, or DR8-mtDRBD. Statistical significances were assessed by a two-tailed unpaired Student’s t test

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