A549, HepG2, HAP1, and 634T WT, ACLY and/or ACSS2-KO cell lines were lysed in mammalian protein extraction reagent (M-PER) buffer (Thermo Fisher Scientific, 78501) with 1× protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific, 23225). Fifteen micrograms of protein was loaded and separated using 4 to 15% Mini-PROTEAN TGX Precast SDS-PAGE Gels (Bio-Rad, #4561086). Samples were then transferred to nitrocellulose membranes for immunoblotting. Total acetylated-lysine (Cell Signaling Technology, 9441), β-actin (Sigma-Aldrich, A5441), ACLY (Cell Signaling Technology, 13390), ACSS2 (Cell Signaling Technology, 3658), and PEX5(D7V4D) (Cell Signaling Technology, 83020) antibodies were used to probe their respective targets. Anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Millipore, AP132P) were used for imaging.
Total acetylated lysine
Manufactured by Cell Signaling Technology
Total acetylated-lysine is a laboratory product that measures the overall level of acetylation on lysine residues within a sample. It provides a quantitative assessment of global protein acetylation status without specific targeting of individual acetylation sites.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Acss2, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ap132p, by Merck Group (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Acss2, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast sds page gel, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Pthr172 ampk, by Cell Signaling Technology (1 mentions)
Atg14, by MBL Life Science (1 mentions)
2 protocols using total acetylated lysine
1
Acetylome Analysis of ACLY and ACSS2 KOs
2
Protein Analysis of Liver Lysates
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The WES capillary electrophoresis system (ProteinSimple, San Jose, CA) was used following standard manufacturer protocols to assess protein levels on total liver lysates. Compass for SW (v4.0.0; ProteinSimple) was used to assess the area under each peak using the Gaussian fit method. The following antibodies and dilutions were used from the respective manufacturers: PDH (1:50; #2784; Cell Signaling, Danvers, MA); PDH p-Ser300 (1:50; AP1064; Sigma-Aldrich); ACLY (1:65; #4332; Cell Signaling); Total Acetylated Lysine (1:65; #9441; Cell Signaling); AMPK (1:50; #2603; Cell Signaling); AMPK p-Thr172 (1:50; #2535; Cell Signaling); ATG14 (1:50; PD026MS; MBL International, Woburn, MA); ATG14 p-Ser29 (1:50; #92340; Cell Signaling).
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