Anti-IRF4 antibody [EP5699]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EP5699] (AB133590)

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IRF4 antibody [EP5699] (AB133590)

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.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-IRF4 antibody [EP5699] (AB133590)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-IRF4 antibody [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).

• IP

Lab

Immunoprecipitation - Anti-IRF4 antibody [EP5699] (AB133590)

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)

All lanes:

Immunoprecipitation - Anti-IRF4 antibody [EP5699] (ab133590)

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Unknown

Western blot - Anti-IRF4 antibody [EP5699] (AB133590)

All lanes:

Western blot - Anti-IRF4 antibody [EP5699] (ab133590) at 1/10000 dilution

Lane 1:

Namalwa cell lysate at 10 µg

Lane 2:

HuT78 cell lysate at 10 µg

Lane 3:

IM9 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 52 kDa

false