Western blot analysis of extracts from KARPAS-620 (human) and A20 (mouse) cells using IRF-4 Antibody #4964 and IRF-4 (P173) Antibody #4948.
Western blot analysis of extracts from SR, KARPAS-620 and Ramos cells using IRF-4 (P173) Antibody.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
IRF-4 (P173) Antibody detects endogenous levels of total IRF-4 protein. This antibody does not cross-react with other family members at physiological levels.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues around Pro173 of human IRF-4 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Interferon regulatory factors (IRFs) comprise a family of transcription factors that function within the Jak/Stat pathway to regulate interferon (IFN) and IFN-inducible gene expression in response to viral infection (1). IRFs play an important role in pathogen defense, autoimmunity, lymphocyte development, cell growth, and susceptibility to transformation. The IRF family includes nine members: IRF-1, IRF-2, IRF-9/ISGF3γ, IRF-3, IRF-4 (Pip/LSIRF/ICSAT), IRF-5, IRF-6, IRF-7, and IRF-8/ICSBP. All IRF proteins share homology in their amino-terminal DNA-binding domains. IRF family members regulate transcription through interactions with proteins that share similar DNA-binding motifs, such as IFN-stimulated response elements (ISRE), IFN consensus sequences (ICS), and IFN regulatory elements (IRF-E) (2).
IRF-4 was independently cloned by three groups and demonstrated to have roles in different contexts of lymphoid regulation (3-5). First, IRF-4 (Pip) was found to associate with PU.1, a member of the ETS family specific to hematopoietic cells, and to regulate the expression of B cell-specific genes (3). Second, it was characterized as a lymphoid-specific member of the IRF family (LSIRF) and able to bind to ISRE (4). Third, it was identified in activated T cells as a factor that binds to the promoter of the interleukin-5 gene (ICSAT), and shown to repress gene activation induced by IFN (5). IRF-4 is expressed in all stages of B cell development and in mature T cells, and is inducible in primary lymphocytes by antigen mimetic stimuli such as Concavalin A, CD3 crosslinking, anti-IgM and PMA treatment (4,5). Mice deficient in IRF-4 show normal distribution of B and T lymphocytes at 4 to 5 weeks, but later develop progressive generalized lymphadenopathy, suggesting a role for IRF-4 in the function and homeostasis of mature B and T lymphocytes (6).
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