Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing full-length Myc/DDK-tagged mouse IRF-7 protein (mIRF-7-Myc/DDK; +) or GST-tagged human IKK-ε protein (hIKK-ε-GST; +), using Phospho-IRF-7 (Ser437/438) (D6M2I) Rabbit mAb (Mouse Specific). A human specific background band is detected at 40 kDa in 293T cellsLearn more about how we get our images.
Western blot analysis of extracts from Raw 264.7 cells, untreated (-) or transfected with poly(deoxyadenylic-deoxythymidylic) acid (dA:dT, 5 μg/ml, overnight; +), using Phospho-IRF-7 (Ser437/438) (D6M2I) Rabbit mAb (Mouse Specific) (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).Learn more about how we get our images.
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
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Phospho-IRF-7 (Ser437/438) (D6M2I) Rabbit mAb (Mouse Specific) recognizes endogenous levels of mouse IRF-7 protein when dually phosphorylated at Ser437 and Ser438. This antibody has a preference for Ser437 and can recognize single phosphorylation at this site.
Monoclonal antibody is produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser437/438 of mosue IRF-7 protein.
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-7, which is functionally similar to IRF-3, is preferentially expressed in lymphoid cells and induced by virus, LPS, and IFN-α (3-5). IRF-7 plays an essential role in the induction of type I interferon in response to viral infection (6-8). Like IRF-3, IRF-7 is regulated at multiple serine phosphorylation sites near its carboxyl terminus, which are required for nuclear translocation, DNA binding, and transcriptional activity (9-11).
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