The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tag USP28 or FLAG-tag LSD1 were used as substrates for protein deacetylation assay. Pull-down of IgG was used as negative control. 0.25 µg of recombinant human GST-tagged HDAC5 protein (Creative BioMart, NY, NY) was mixed with 30 µl immunoprecipitates or 1.5 µg bulk histone at 37°C for 6h in a buffer containing 40 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 50 mM NaCl, 2 mM KCl, 0.5mM DTT, 1mM EDTA and protease inhibitor. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody (EMD Millipore, Billerica, MA). FLAG-tagged USP28 or LSD1 and bulk histone were probed with anti-FLAG antibody or H3 antibody as loading control. Inactive HDAC5-GST protein was used as negative control by heating recombinant protein at 95°C for 5 min. In vivo protein acetylation assay was performed using cell lysate of MDA-MB-231 cell transfected with scramble and HDAC5 siRNAs. LSD1 or IgG antibodies were added to cell lysate. Protein G-plus agarose beads (Santa Cruz) were collected and subjected to immunoblotting with anti-acetyl lysine or LSD1 antibodies.
Anti acetyl lysine antibody
Manufactured by Merck Group
The Anti-acetyl lysine antibody is a laboratory reagent used in biochemical and molecular biology research. It is designed to specifically recognize and bind to acetylated lysine residues within proteins. This antibody can be used in various analytical techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the acetylation status of proteins.
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2 protocols using anti acetyl lysine antibody
1
Deacetylation Assay of USP28 and LSD1 Complexes
2
Deacetylation Assay of USP28 and LSD1 Complexes
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The immunoprecipitates of FLAG-M2 agarose from MDA-MB-231 cells overexpressing FLAG-tag USP28 or FLAG-tag LSD1 were used as substrates for protein deacetylation assay. Pull-down of IgG was used as negative control. 0.25 µg of recombinant human GST-tagged HDAC5 protein (Creative BioMart, NY, NY) was mixed with 30 µl immunoprecipitates or 1.5 µg bulk histone at 37°C for 6h in a buffer containing 40 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 50 mM NaCl, 2 mM KCl, 0.5mM DTT, 1mM EDTA and protease inhibitor. The reactions were then subjected to immunoblots with anti-acetyl lysine antibody (EMD Millipore, Billerica, MA). FLAG-tagged USP28 or LSD1 and bulk histone were probed with anti-FLAG antibody or H3 antibody as loading control. Inactive HDAC5-GST protein was used as negative control by heating recombinant protein at 95°C for 5 min. In vivo protein acetylation assay was performed using cell lysate of MDA-MB-231 cell transfected with scramble and HDAC5 siRNAs. LSD1 or IgG antibodies were added to cell lysate. Protein G-plus agarose beads (Santa Cruz) were collected and subjected to immunoblotting with anti-acetyl lysine or LSD1 antibodies.
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