PDC from porcine heart tissue (Sigma, catalog no. P7032) was concentrated by centrifugation at 135,000 X g for 2 h in 100 mM potassium phosphate, pH 7.5, 0.05% lauryl maltoside, 2.5 mM EDTA, and 30% glycerol as described before51 (link). Desuccinylation reactions were carried out on 7 μg of purified porcine heart PDC, using 2 μg of purified Sirt5 or Sirt5H158Y in a final reaction volume of 20 μL in presence of 25 mM Tris-Cl, pH 8.0, 200 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.1 % PEG 8000, and 3.125 mM NAD+ at 37 °C for 30 min. During incubation with compounds, tubes were occasionally agitated. Reactions were analyzed by immunoblotting with a succinyl-lysine antibody (PTM Biolabs). After analysis, the membrane was stripped and re-probed with PDHA1 antibody (Abcam) and Sirt5 antibody (Cell Signaling Technology).
Pdha1 antibody
Manufactured by Abcam
PDHA1 antibody is a primary antibody that specifically binds to the PDHA1 (Pyruvate dehydrogenase E1 component subunit alpha, somatic form) protein. PDHA1 is a key enzyme involved in the conversion of pyruvate to acetyl-CoA, which is an essential step in cellular energy metabolism. This antibody can be used for the detection and analysis of PDHA1 in various biological samples.
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Lab products found in correlation
Succinyl lysine antibody, by PTM Biolabs (1 mentions)
Sirt5 antibody, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 donkey anti mouse igg a21202, by Thermo Fisher Scientific (1 mentions)
Lc3 antibody, by Cell Signaling Technology (1 mentions)
Tcs spe, by Leica (1 mentions)
Draq5, by Biostatus (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
2 protocols using pdha1 antibody
1
Porcine Heart PDC Desuccinylation by Sirt5
2
Immunofluorescence Assay of Autophagy
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Hep3B cells were seeded at a density of 50,000 cells/well in 12-well plates on 10 mm round coverslips. After treatments, cells were rinsed with PBS, fixed with 4% PFA for 15 min, washed with PBS kept, blocked in a solution of 1% fatty acid free BSA, 0.1% saponin and 0.5% glycine in PBS for 20 min at RT and incubated with primary antibodies overnight at 4 °C inside a dark chamber (LC3 antibody, #2775S, Cell Signaling Technology®, 1/300, rabbit; PDHA1 antibody, ab110330, Abcam, 1/200, mouse) in 0.05% saponin and Dako Antibody Diluent with Background Reducing Components as solvent. After washing, samples were incubated with secondary antibodies (1 h at RT, anti-rabbit Cy3, 1/300; Alexa Fluor 488 donkey anti-mouse IgG A21202 Invitrogen, 1/300) and acid nucleic marker DRAQ5TM (DR50200, BioStatus, Leicestershire, UK), washed and mounted in 5 µL of Fluoromount-G® (0100-01, Southern Biotech, Birmingham, AL, USA). Pictures, ten random fields per sample, were taken at the confocal microscope Leica TCS SPE with the 60× oil objective.
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