PKC activity was examined by a non-radioactive method according to a previously published study [36 (link)]. Briefly, hippocampal tissues were lysed in 1000 μl PKC extraction buffer (1 μg/ml leupeptin [Sigma-Aldrich], 1 μg/ml aprotinin [Sigma-Aldrich], 25 mM Tris [Sigma-Aldrich], 10 mM mercaptoethanol [Sigma-Aldrich], 10 mM β-mercaptoethanol [Sigma-Aldrich], 0.05% Triton X-100 [Sigma-Aldrich], 0.5 mM EDTA [Sangon, Shanghai, China], and 0.5 mM EGTA [Sangon]). Equal amounts of hippocampal tissue proteins were utilized in each PKC reaction according to the protocol of the PepTag® non-Radioactive Protein Kinase C detection kit (Promega, Madison, WI, USA). The hippocampal tissue samples were then incubated with a fluorescent, positively-charged, PKC-specific peptide (C1 peptide, 0.4 μg/μl) for 30 min and separated on 0.8% agarose gels. The phosphorylated, negatively-charged peptide was separated from non-phosphorylated, positively-charged peptide and visualized under UV light. The bands were quantified by densitometry and normalized to the controls.
Ethylene glycol tetraacetic acid (egta)
Manufactured by Sangon
Sourced in China
EGTA is a chemical compound commonly used as a chelating agent in laboratory settings. It has the ability to bind to and sequester metal ions, particularly calcium ions. This property makes EGTA useful for various applications in biochemistry and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Mercaptoethanol, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Sangon (1 mentions)
Triton x 100, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Anti his antibody, by Transgene (1 mentions)
Anti gst antibody, by Transgene (1 mentions)
2 protocols using ethylene glycol tetraacetic acid (egta)
1
Non-Radioactive Quantification of PKC Activity
2
Protein-Protein Interaction Assay
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Pull-down assays were carried out as previously described (Yan et al., 2018) . Briefly, 30-μg bait protein (MdCRF4-His, MdCRF4 H , MdCRF4 N , MdCRF4 CBD , MdCRF4DD-His, or MdCRF4AA-His) was captured with Ni-NAT resin (Transgene, Beijing China; https://www.transgen.com.cn), washed 5 times, then incubated with 10-μg prey protein (MdCaM2-GST, MdXBAT31-GST, or empty GST) at 4°C for 1 h in RB buffer pH 7.8, 0.1% [v/v] glycerol, and 20-mM β-mercaptoethanol). After washing 5 times with the RB buffer to remove nonspecifically bound proteins, the precipitated prey proteins were released by boiling in sodium dodecyl sulfate (SDS) sample buffer at 100°C for 5 min before detection by immunoblot analysis using an anti-His antibody (Cat. no. HT501, Transgen, China). The relative input of the prey proteins was detected by immunoblot analysis using an anti-GST antibody (Cat. no. HT601, Transgen, China). For Ca 2+mediated interactions between MdCRF4 and MdCaM2 assays, 1-mM CaCl 2 or 5-mM EGTA (Sangon Biotech, China) was added to the RB buffer.
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