重组Anti-KAP1抗体[EPR5249] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation :

Overlay histogram showing HeLa cells stained with ab109545 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab190545, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation :

ab109545, at a 1/100 dilution, staining KAP1 in HeLa cells

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation :

ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human colon tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation :

ab109545, at a 1/100 dilution, staining KAP1 in formalin-fixed, paraffin-embedded Human kidney tissue.

Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

KAP1 was immunoprecipitated from 0.35 mg A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 μg with ab109545 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab109545 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) cell lysate 10 μg

Lane 2 : ab109545 IP in A431 cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab109545 in A431 cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 32 seconds

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab109545).

All lanes:

Immunoprecipitation - Anti-KAP1 antibody [EPR5249] (<a href='/products/primary-antibodies/kap1-antibody-epr5249-ab109545'>ab109545</a>)

Predicted band size: 88 kDa

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• WB

Lab

Western blot - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation :

Blocking/diluting buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (ab247904) at 1/10000 dilution

Lane 1:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 15 µg

Lane 2:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 88 kDa

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Exposure time: 10s

• WB

Lab

Western blot - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation.

Western blot : Anti-KAP1 antibody [EPR5249] ab109545 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 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-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-KAP1 antibody [EPR5249] (<a href='/products/primary-antibodies/kap1-antibody-epr5249-ab109545'>ab109545</a>) at 1/1000 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-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 89 kDa

Observed band size: 110 kDa,37 kDa

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• WB

Lab

Western blot - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (AB247904)

This data was developed using ab109545, the same antibody clone in a different buffer formulation :

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : HAP1 + DMSO whole cell lysate (20 μg)
Lane 3 : HAP1 + Blaomycin whole cell lysate (20 μg)
Lane 4 : TRIM28 knockout HAP1 whole cell lysate (20 μg)
Lane 5 : TRIM28 knockout HAP1 + DMSO whole cell lysate (20 μg)
Lane 6 : TRIM28 knockout HAP1 + Blaomycin whole cell lysate (20 μg)
Lane 7 : HeLa + DMSO whole cell lysate (20 μg)
Lane 8 : HeLa + Blaomycin whole cell lysate (20 μg)

Lanes 1 - 8 : Merged signal (red and green). Green - ab109545 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab109545 was shown to specifically react with KAP1 in wild type cells as signal was lost in KAP1 knockout cells. Wild-type and KAP1 knockout samples were subjected to SDS-PAGE. ab109545 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-KAP1 antibody [EPR5249] - BSA and Azide free (ab247904)

Predicted band size: 88 kDa

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