Rabbit anti-acetyllysine antibody (Cell Signaling Technology, Beverly, MA) was used at 1:2500. Rabbit anti-LCAD, anti-very long-chain acyl-CoA dehydrogenase (VLCAD), and anti-electron transferring flavoprotein (ETF) antisera (kind gifts of Dr. Jerry Vockley, Children’s Hospital of Pittsburgh) were used at 1:2500. Note that the anti-ETF antiserum recognizes the α and β subunits of ETF, producing two bands upon immunoblot. For immunoprecipitation, Flag-tagged LCAD and VLCAD proteins were recovered from transiently transfected HEK293 cells using anti-Flag M2 antibody (Sigma). Fractionation of cells into mitochondria and cytosol was conducted exactly as described [18 (link)].
Rabbit anti acetyl lysine antibody
Manufactured by Cell Signaling Technology
Rabbit anti-acetyl lysine antibody is a lab equipment product that recognizes acetylated lysine residues in proteins. It is a tool for detecting and studying protein acetylation, a post-translational modification important in various cellular processes.
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Lab products found in correlation
Anti flag m2 antibody, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Anti cd38 apc, by BD (1 mentions)
Anti cd11b apc, by BD (1 mentions)
Anti acetylated foxo1, by Santa Cruz Biotechnology (1 mentions)
Anti cd90 pe, by BD (1 mentions)
Anti cd38 fitc, by BD (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Annexin 5, by BD (1 mentions)
6 protocols using rabbit anti acetyl lysine antibody
1
Antibody-based Detection of Mitochondrial Proteins
2
Curcumin Modulates Smad2 Acetylation
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H9c2 cells from ATCC were pre-incubated with 25 μM curcumin for 4 hrs prior to being stimulated with 5 ng TGF-β1 for 24 hrs. Total protein was extracted from cells using a lysis buffer. Smad2 was immune-precipitated using a goat polyclonal anti-Smad2/3 antibody. Acetylation of Smad2 was assessed by Western blotting using a rabbit anti-acetyl-lysine antibody (Cell Signaling Technology, Danvers MA).
3
SIRT1 Regulation of Ku70 Acetylation
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Cells were harvested and lysed in lysis buffer for protein extraction followed by the determination of protein concentration, and the supernatant was used for Western blotting. Proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies (Rabbit polyclonal anti-human SIRT1 [Epitomics], 1:1000; rabbit anti-human Ku70 [Epitomics], 1:1000; rabbit polyclonal anti-acetylated p53 [Cell Signaling Technology], 1:1000; rabbit polyclonal anti-acetylated FOXO1 [Santa Cruz Biotechnology], 1:1000; β-actin [Cell Signaling Technology], 1:1000) followed by incubation with secondary antibodies conjugated to horseradish peroxidase (HRP-donkey-anti-rabbit). Signal development was performed with an ECL kit. The experiment was performed three times. To analyze Ku70 acetylation, Ku70 was pulled down from total cell lysate with conjugated anti-Ku70-agarose beads (Santa Cruz Biotechnology), followed by acetylation detection with rabbit anti-acetyl lysine antibody (Cell Signaling Technology). Moreover, in order to analyze the direct interaction between SIRT1 and Ku70, SIRT1 or Ku70 was pulled down from total cell lysate with conjugated anti-SIRT1 or Ku70-agarose beads (Santa Cruz Biotechnology), followed by Ku70 or SIRT1 detection with rabbit anti-Ku70 or SIRT1 antibody (Cell Signaling Technology).
4
Fluorescence-Activated Cell Sorting and Analysis
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For fluorescence-activated cell sorting or analysis, half million cells were stained with fluorophore conjugated antibodies in a buffer (0.5% bovine serum albumin in phosphate-buffered saline) for 15 min on ice. Antibodies used for analysis and sorting were: APC-anti CD38, FITC-anti CD38, PE-anti CD90, APC-anti-CD11b (BD Pharmingen), and rabbit polyclonal anti-acetylated FOXO1 (Santa Cruz Biotech). Apoptosis was analyzed by annexin-V (BD Pharmingen) staining. Flow cytometry was performed at the City of Hope Flow Cytometry Core.
For Western blot, rabbit monoclonal anti-human SIRT1 (Epitomics), mouse monoclonal anti-Ku70 (Neomarker) were used. To analyze Ku70 acetylation, we pulled down Ku70 from total cell lysate with anti-Ku70 and protein A/G plus-agarose beads (Santa Cruz) followed by acetylation detection with the rabbit anti-acetyl lysine antibody (Cell Signaling).
For Western blot, rabbit monoclonal anti-human SIRT1 (Epitomics), mouse monoclonal anti-Ku70 (Neomarker) were used. To analyze Ku70 acetylation, we pulled down Ku70 from total cell lysate with anti-Ku70 and protein A/G plus-agarose beads (Santa Cruz) followed by acetylation detection with the rabbit anti-acetyl lysine antibody (Cell Signaling).
5
SIRT1 Interaction and Acetylation Analysis
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Cells were lysed in lysis buffer followed by the determination of protein concentration. In order to analyze the direct interaction between SIRT1 and autophagy-related genes LC3B or BECN1, SIRT1 or LC3B, or BECN1 was pulled down from total cell lysate with conjugated anti-SIRT1 or LC3B or BECN1-agarose beads (Santa Cruz Biotechnology), followed by LC3B or BECN1 or SIRT1 detection with rabbit anti-LC3B or BECN1 or SIRT1 antibody (Cell Signaling Technology). To analyze LC3B and BECN1 acetylation, LC3B or BECN1 was pulled down from total cell lysate with conjugated anti-LC3B or BECN1-agarose beads (Santa Cruz Biotechnology), followed by acetylation detection with rabbit anti-acetyl lysine antibody (Cell Signaling Technology). The experiments were performed three times.
6
Detecting Protein Modifications by Western Blot
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VLCAD was detected using either mouse monoclonal anti-poly-His antibody (Sigma, St. Louis, MO) at a 1:5000 dilution or rabbit anti-VLCAD antiserum at 1:2500. Lysine acetylation and succinylation were detected using rabbit anti-acetyllysine antibody (Cell Signaling Technology, Beverly, MA) and anti-succinyllysine antibodies [5 (link)], respectively, both at 1:1000 dilution. After incubation with HRP-conjugated secondary antibodies (1:5000) blots were visualized with chemiluminescence.
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