Anti-KAP1 抗体 [20C1]

• WB

Supplier Data

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

Western blot analysis was performed on nuclear enriched extracts (30 μg lysate) of cells treated with Hydrogen peroxide (100 μm Hydrogen peroxide for 10 min) or untreated. The blots were probed with ab22553 at 1 μg/mL and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate. A 110 kDa band corresponding to KAP1 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex^® NuPAGE^® 4-12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex^® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot^® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

All lanes:

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

Lanes 1 - 2:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 30 µg

Lane 3:

A549 (Human lung carcinoma cell line) whole cell lysate at 30 µg

Lane 4:

HEL 92.1.7 whole cell lysate at 30 µg

Lane 5:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate at 30 µg

Lane 6:

Caco-2 (Human colorectal adenocarcinoma cell line) whole cell lysate at 30 µg

Predicted band size: 88 kDa

Observed band size: 110 kDa

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• 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

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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