Western blot analysis of extracts from various cell lines using NOP2 (L221) 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
NOP2 (L221) Antibody recognizes endogenous levels of total NOP2 protein.Species Reactivity:
Human, Mouse, Rat
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu221 of human NOP2 protein. Antibodies are purified by peptide affinity chromatography.
Chemical modifications of RNA regulate many cellular processes. One particular RNA modification, 5-methylcytosine (m5C), regulates ribosome assembly, translation, and RNA stability (1). In eukaryotes, this modification is added to RNA by the DNA methyltransferase homologue 2 protein (DNMT2; also known as TRDMT1), and also by members of the NOL1/NOP2/SUN domain (NSUN) family of proteins. NSUN proteins are putative S-adenosylmethionine (SAM)-dependent methyltransferases that carry out their enzymatic activity by utilizing two cysteine residues in their active sites (2). There are currently seven known members of this family, including NOP2 (NSUN1) and NSUN2-7.
NOP2, also known as NSUN1, is an 89 kDa member of the NSUN protein family that specifically targets and methylates 28S rRNA (3). In humans, methylation of C4413 in the 28S rRNA by NOP2 is thought to increase stability of the ribosome. NOP2 is strongly overexpressed in many cancers, including colorectal and lung cancer, where it may contribute to tumorigenesis by recruiting telomerase to the cyclin D1 promoter and activating gene expression (4-6). NOP2 also interacts with BRD4 and RNA polymerase II, suggesting additional roles for NOP2 in regulating transcription (1,7).
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