Western blot analysis of extracts from various tissues and cell lines using PME-1 (8A6-F8) Mouse mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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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.
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: 262
PME-1 (8A6-F8) Mouse mAb recognizes endogenous levels of total PME-1 protein.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Monoclonal antibody is produced by immunizing animals with recombinant full-length mouse PME-1 protein.
Protein phosphatase methylesterase 1 (PME-1) is an evolutionarily conserved enzyme that demethylates phosphatases (1). Post-translational modification (PTMs) of proteins is a cellular mechanism that increases the functional diversity of the proteome. Several forms of PTMs exist, including methylation and phosphorylation, the covalent addition of a methyl or phosphate group, respectively, to specific amino acids within a protein. In addition to enzymes that catalyze the addition of methyl groups or phosphates to proteins, specific enzymes that remove PTMs exist to provide an additional level of cellular regulation; methyl and phosphate PTMs are removed by methylesterases and phosphatases, respectively. Phosphoprotein phosphatase 2a (PP2A) is an essential serine/threonine phosphatase that, as part of various signal transduction pathways, regulates many fundamental cellular processes, including DNA replication, transcription, translation, metabolism, cell cycle progression, cell division, apoptosis, and development (2-4). PP2A function is regulated, in part, by phospho- and methyl modification of its catalytic subunit. PP2A is methylated at the carboxyl group of the C-terminal Leucine 309 residue by leucine carboxyl methyltransferase (LCMT). Methylation of PP2A alters its cellular localization and its ability to interact with its regulatory subunits and substrates (5-8). PP2A is demethylated by PME-1 (9,10). PME-1 KO mice are post-natal lethal, and KO tissue exhibit altered PP2A activity and phospho-proteomic profile, consistent with a critical role PME-1 plays in regulating PP2A function (11). Dysregulated PP2A activity is linked to several diseases, including certain cancers and neurodegenerative diseases like Alzheimer’s disease, suggesting that PME-1 could be the target of therapeutic intervention (12-14).
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