Western blot analysis of extracts from 293T cells untreated (-) or treated with Sodium-Propionate (Na-Prop, 50 mM; 16 hours), using Propionyl-Lysine [Prop-K] (D3A9R) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).Learn more about how we get our images.
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
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.
Propionyl-Lysine (D3A9R) Rabbit mAb recognizes endogenous levels of proteins only when propionylated at a lysine residue. This antibody does not cross-react with other lysine modifications.
All Species Expected
Monoclonal antibody is produced by immunizing animals with a synthetic peptide library containing propionyl-lysine.
Lysine is subject to a wide array of regulatory post-translational modifications due to its positively charged ε-amino group side chain. The most prevalent of these are ubiquitination and acetylation, which are highly conserved among prokaryotes and eukaryotes (1,2). Acyl group transfer from the metabolic intermediates acetyl-, succinyl-, malonyl-, glutaryl-, butyryl-, propionyl-, and crotonyl-CoA all neutralize lysine’s positive charge and confer structural alterations affecting substrate protein function. Lysine acetylation is catalyzed by histone acetyltransferases, HATs, using acetyl-CoA as a cofactor (3,4). Deacylation is mediated by histone deacetylases, HDACs 1-11, and NAD-dependent Sirtuins 1-7. Some sirtuins have little to no deacetylase activity, suggesting that they are better suited for other acyl lysine substrates (5).
Protein propionyl and butyryl transferase activity has been reported for p300 and CREB-binding protein, two acetyltransferases that can autoacylate as well as target histone proteins and p53 in vitro. Sirt1 (Sir2 in yeast) has been shown to have depropionylase activity and may be a major eukaryotic depropionylase (6,7). In the cytosol, acetyl-CoA carboxylase (ACC) converts acetyl-CoA to Malonyl-CoA and the reverse reaction is catalyzed by Malonyl-CoA decarboxylase (MCD), but in the mitochondria, propionyl-CoA carboxylase takes the role of ACC. Both MCD and ACC are regulated by AMPK, glucose levels, and insulin, underscoring their importance in intermediary metabolism (8).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered trademark of Cell Signaling Technology, Inc. Tween is a registered trademark of ICI Americas, Inc. Use of Cell Signaling Technology (CST) Motif Antibodies within certain methods (e.g., U.S. Patents No. 7,198,896 and 7,300,753) may require a license from CST. For information regarding academic licensing terms please have your technology transfer office contact CST Legal Department at CST_ip@cellsignal.com. For information regarding commercial licensing terms please contact CST Pharma Services Department at email@example.com.
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