PARKIN phosphorylation was performed by incubating 5 μM HsPARKIN with 0.5 μM GST-TcPINK1, 10 mM ATP, 1x reaction buffer and phosphoUb (14 μM unless stated differently). The reaction was quenched at the given time points with LDS sample buffer and proteins were separated on a NuPAGE 4-12% gradient Bis-Tris gel, transferred on nitrocellulose membrane and detected with anti-pSer65 PARKIN antibody (Abcam cat no. ab154995). Phosphorylation assays of HsPARKIN Ubl domain (aa 1-72) and Ub were performed as described above with 20 μM of HsPARKIN Ubl domain and ubiquitin, respectively. For the Ub phosphorylation assay, the GST-TcPINK1 concentration was increased to 1.5 μM. The reaction was quenched at the given time points with LDS sample buffer and proteins were separated on a 15% SuperSep Phos-tag™ gel (Wako Chemicals) and stained with Instant Blue SafeStain (Expedeon).
Anti pser65 parkin antibody
Manufactured by Abcam
The Anti-pSer65 PARKIN antibody is a laboratory research tool designed to detect the phosphorylated form of the PARKIN protein at serine residue 65. PARKIN is an E3 ubiquitin ligase involved in mitophagy, a process of selectively removing damaged mitochondria from cells. Phosphorylation of PARKIN at serine 65 is an important regulatory mechanism for its activation and recruitment to mitochondria. This antibody can be used to study the phosphorylation state of PARKIN in various cellular and biochemical assays.
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2 protocols using anti pser65 parkin antibody
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PARKIN Phosphorylation by PINK1
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PARKIN Phosphorylation by PINK1
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PARKIN phosphorylation was performed by incubating 5 μM HsPARKIN with 0.5 μM GST-TcPINK1, 10 mM ATP, 1x reaction buffer and phosphoUb (14 μM unless stated differently). The reaction was quenched at the given time points with LDS sample buffer and proteins were separated on a NuPAGE 4-12% gradient Bis-Tris gel, transferred on nitrocellulose membrane and detected with anti-pSer65 PARKIN antibody (Abcam cat no. ab154995). Phosphorylation assays of HsPARKIN Ubl domain (aa 1-72) and Ub were performed as described above with 20 μM of HsPARKIN Ubl domain and ubiquitin, respectively. For the Ub phosphorylation assay, the GST-TcPINK1 concentration was increased to 1.5 μM. The reaction was quenched at the given time points with LDS sample buffer and proteins were separated on a 15% SuperSep Phos-tag™ gel (Wako Chemicals) and stained with Instant Blue SafeStain (Expedeon).
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