|H Mk||Endogenous||42||Rabbit IgG|
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
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.
SMYD3 (D2Q4V) Rabbit mAb recognizes endogenous levels of total SMYD3 protein. This antibody does not cross-react with other SMYD proteins.
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro357 of human SMYD3 protein.
SET and MYND domain containing protein 3 (SMYD3) is a member of the SET domain-containing family of protein methyltransferases and is localized to both the nucleus and cytoplasm (1-3). Several histone substrates have been identified for SMYD3; however, the data is controversial. In one study, SMYD3 has been shown to methylate histone H3 Lys4 (both di- and tri-methylation) and interact with RNA polymerase II to activate transcription (1). A second study has shown that SMYD3 preferentially methylates histone H4 Lys20 and interacts with nuclear receptor corepressor complex (NCOR) to repress transcription (2). A third study has shown that SMYD3 preferentially methylates histone H4 Lys5 (mono-, di-, and tri-methylation) (3). In addition, SMYD3 has been shown to methylate the endothelial growth factor receptor 1 (VEGFR1) on Lys831 and stimulate its kinase activity (4). Regardless of the preferred protein substrates, it is clear that SMYD3 functions as an oncogene. Research studies have shown SMYD3 is highly over-expressed in liver, breast, and rectal carcinomas. Over-expression of SMYD3 in multiple cell lines enhances proliferation, adhesion, and migration, while reduced expression results in significant suppression of cell growth (1,5-10). In addition, multiple cancer cell lines express both full length SMYD3 and a cleaved form of SMYD3 lacking the N-terminal 34 amino acids, and the cleaved form shows increased methyltransferase activity toward histone H3 (11).
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|12859S||100 µl (10 western blots)||$ 255.0|