重组Anti-IRF4抗体[EP5699] (ab133590)

Immunocytochemistry/ Immunofluorescence analysis of IM-9 (human multiple myeloma B lymphoblast) labeling IRF4 with ab133590 at 1/500 dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% TritonX-100. DAPI was used as nuclear counterstain. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) at 1/200 was used as the counterstain antibody.

Confocal image showing mainly nuclear staining in IM-9 cell line.
Negative control: 293T (PMID: 23804715）
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

IRF4 was immunoprecipitated from 0.35 mg IM-9 (Human multiple myeloma B Lymphoblast) whole cell lysate 10 µg with 133590 at 1/20 dilution (1µg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: IM-9 (Human multiple myeloma B Lymphoblast) whole cell lysate 10 µg

Lane 2: ab133590 IP in IM-9 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab133590 in IM-9 whole cell lysate

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

Lower bands could be ubiquitin mediated protein degradation. (PMID: 23658517)

Overlay histogram showing Ramos cells stained with ab133590 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab133590, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemical analysis of paraffin embedded Human lymphoma tissue labelling IRF4, using ab133590 at a dilution of 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded Human tonsil tissue labelling IRF4, using ab133590 at a dilution of 1/250.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.