5/31/2023 0 Comments Akta prime plus softwarePerform the SDS denaturation test (SD-test) that was developed for the analysis of the source of a ligand-induced fluorescence change that exceeds ☒0% of the fluorescence average. Structure of human LARS1 methyl-Leu-AMS synĪmicon Ultra-15 Centrifugal Filter 30kDa cutoffĢ2 mm × 0.22 mm Siliconized square cover slides His-Tag Labeling Kit RED-tris-NTA 2 nd Generation To begin, we needed to generate the expression plasmid by performing DNA cloning and transformation.Ĭhemicals, peptides, and recombinant proteins We present the crystallization and structure determination of LARS1 with leucine, Leu-AMS, and ATP. In this protocol, we describe a procedure for the purification and the reductive methylation ( Walter et al., 2006 Kobayashi et al., 1999)) of LARS1, and measurements by MST and DSF. We also measured the binding affinity of leucine for LARS1 using MicroScale Thermophoresis (MST) and checked T i values using differential scanning fluorimetry (DSF). Recently, we were able to obtain crystals of LARS1 complexed with leucine, Leu-AMS, and ATP, respectively, and improved crystal behavior by reductive methylation of lysine and post-crystallization soaking & cooling in cryoprotectants at −20☌. LARS1 is a multidomain and flexible protein ( Figure 1) therefore, the X-ray structure of LARS1 has not been solved for a long time. ![]() LARS1 protein is composed of 1,176 amino acid residues, and contains a catalytic domain, an editing domain, a dynamic C-terminal domain composed of a RagD-binding domain (RBD), LVβ, and UNE-L domains.
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