重组Anti-IKB beta抗体[EPR5037]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB beta antibody [EPR5037] (AB109509)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue using unpurified ab109509 at a dilution of 1/100.

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IKB beta antibody [EPR5037] (AB109509)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling IKB beta with purified ab109509 at 1 : 500 dilution (0.20 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IKB beta antibody [EPR5037] (AB109509)

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling IKB beta with purified ab109509 at 1/20 dilution (10 μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IKB beta antibody [EPR5037] (AB109509)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling IKB beta with purified ab109509 at 1 : 50 dilution (2.0 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IP

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Immunoprecipitation - Anti-IKB beta antibody [EPR5037] (AB109509)

ab109509 (purified) at 1 : 20 dilution (1 μg) immunoprecipitating IKB beta in HeLa whole cell lysate.

Lane 1 (input) : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+) : ab109509 & HeLa whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109509 in HeLa whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1 : 1000 dilution.

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-IKB beta antibody [EPR5037] (ab109509)

Predicted band size: 37 kDa

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• WB

Lab

Western blot - Anti-IKB beta antibody [EPR5037] (AB109509)

Lane 1 : Wild type HAP1 whole cell lysate (20 μg)
Lane 2 : IKB beta knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - unpurified ab109509 observed at 45 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab109509 was shown to specifically react with IKB beta in wild type cells as signal was lost in IKB beta knockout cells. Wild-type and IKB beta knockout samples were subjected to SDS-PAGE. ab109509 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 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-IKB beta antibody [EPR5037] (ab109509)

Predicted band size: 37 kDa

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• WB

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Western blot - Anti-IKB beta antibody [EPR5037] (AB109509)

All lanes:

Western blot - Anti-IKB beta antibody [EPR5037] (ab109509) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

THP-1 cell lysate at 10 µg

Lane 4:

MCF7 cell lysate at 10 µg

Predicted band size: 37 kDa

Observed band size: 48 kDa

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• WB

Lab

Western blot - Anti-IKB beta antibody [EPR5037] (AB109509)

All lanes:

Western blot - Anti-IKB beta antibody [EPR5037] (ab109509) at 1/1000 dilution

All lanes:

HeLa (Human cervix 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: 37 kDa

Observed band size: 48 kDa

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