Western blot analysis of extracts from HeLa cells, untreated (-) or treated with hIFN-α1 #8927 (+), and Hep G2 and Vero cells, using ADAR1 (D7E2M) Rabbit mAb.
|MW (kDa)||110, 150|
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.
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
ADAR1 (D7E2M) Rabbit mAb recognizes endogenous levels of total ADAR1 protein.Species Reactivity:
Human, MonkeySpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Trp454 of human ADAR1 protein.
Post-transcriptional processing of RNAs such as RNA editing is an important mechanism by which diversity in RNA and protein is achieved that is not otherwise encoded by the genome (1,2). The most common form of RNA editing is the conversion of adenosine (A) into inosine (I) on double stranded RNA by the adenosine deaminase acting on RNA (ADAR) family of proteins (1-3). Since inosine base pairs with cytidine, it is interpreted as a guanosine by the splicing and translational machinery leading to alteration in the protein sequence, as well as splicing isoforms being generated (1,4-6). A-to-I editing can also influence RNA sequence recognition by RNA binding proteins and non-coding RNA, such as miRNAs, affecting subsequent RNA processing, stability, and protein expression levels (2).
ADAR1 is ubiquitously expressed with two known isoforms ADAR1L (p150) and ADAR1S (p110) resulting from transcription using alternative promoters and start codons. ADAR1S is constitutively expressed in the nucleus, while ADAR1L is interferon-inducible and present in both the nucleus and the cytoplasm. The induction of ADAR1L in response to cellular stress and viral infection suggests a role for RNA editing in the innate immune response (1,7). In addition, ADAR1 is essential in mammalian development, particularly in hematopoiesis and suppression of interferon signaling to protect hematopoietic stem cells from destruction in the fetal liver and the adult bone marrow (8,9).
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