Western blot analysis of extracts from various cell lines, including NIH/3T3 RPL29 wild-type (WT) and NIH/3T3 RPL29 knockout (KO), using Di-Methyl-RPL29 (Lys5) (D8T9P) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
Peptide dot blot analysis demonstrating Di-Methyl-RPL29 (Lys5) (D8T9P) Rabbit mAb specificity. Antibody binding to pre-coated RPL29 peptides is shown using Di-Methyl-RPL29 (Lys5) (D8T9P) Rabbit mAb. As expected, the Di-Methyl-RPL29 (Lys5) (D8T9P) Rabbit mAb binds to RPL29 peptide when dimethylated at Lys5. The antibody also shows slight binding to RPL29 peptide that is monomethylated at Lys5.
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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
Di-Methyl-RPL29 (Lys5) (D8T9P) Rabbit mAb recognizes endogenous levels of RPL29 protein only when methylated at Lys5. This antibody shows slight cross-reactivity to mono-methyl-RPL29 Lys5.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic methyl peptide corresponding to residues surrounding Lys5 of human RPL29 protein.
SET7/SET9 is a member of the SET domain-containing family, and can specifically methylate Lys4 on histone H3 (1). Like most other lysine-directed histone methyltransferases, it contains a conserved catalytic SET domain originally identified in the Drosophila Su(var)3-9, Enhancer of zeste and Trithorax proteins. Histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development (2,3). Methylation of histone H3 Lys4 enhances transcriptional activation by coordinating the recruitment of BPTF, a component of the NURF chromatin remodeling complex, and WDR5, a component of multiple histone methyltransferase complexes (4,5). In addition, methylation of lysine 4 blocks transcriptional repression by inhibiting the binding of the NURD histone deacetylation complex to the amino-terminal tail of histone H3 and interfering with SUV39H1-mediated methylation of histone H3 Lys9 (1). SET7/SET9 is highly active on free histone H3, but only very weakly methylates H3 within nucleosomes (1). Besides histones, SET7/SET9 also methylates Lys189 of the TAF10, a member of the TFIID transcription factor complex, and Lys372 of the p53 tumor suppressor protein (6,7). Methylation of TAF10 stimulates transcription in a promoter-specific manner by increasing the affinity of TAF10 for RNA polymerase II, which may potentiate pre-initiation complex formation (6). Methylation of p53 at Lys372 increases protein stability and leads to upregulation of target genes such as p21. Thus the loss of SET7/SET9 may represent another mechanism for the inactivation of p53 in human cancers (7).
Ribosomal protein L29 (RPL29) is a ubiquitously expressed protein subunit of the cytoplasmic eukaryotic large 60S ribosomal subunit that functions in the translation of RNA to protein. RPL29 is a non-histone substrate of the SET7/SET9 protein methyltransferase and is exclusively mono- and di-methylated on lysine 5 by SET7/SET9. In addition, RPL29 lysine 5 is demethylated by the LSD1 protein demethylase (8). This antibody provides a specific readout for SET7/SET9 methyltransferase and LSD1 demethylase activities.
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