研修計劃要點(英文)(存儲版)
【正文】 on a protein ASepharose column. 2. Rebinant Proteins Sun1 was initially isolated from human brain. SUN will be PCRamplified from the Sun1 cDNA and directly cloned in TOPO/V5His and pET21 vectors. Rebinant pET vectors will be transformed into BL21(DE3)plysS host which is ideally stringent to express membrane proteins. Verify if rebinant protein is expressed in inclusion bodies and purify it by Ni column. 3. Gel electrophoresis and Immunoblotting Test the dilution first and then use the Sun1 serum as the primary antibody. 4. Proteinase K Protection Assays The Sun1 cDNA cloned in the [ 35 S]methionine in the absence or the presence of caninepancreatic microsomal membranes. Aliquots of the synthesis reaction will be digested for 30 min on ice with proteinase K. Proteinase K will be inhibited with PMSF, and the samples will be added directly to boiling Laemmli buffer. Duplicate samples will be transferred to a nitrocellulose membrane, and the radiolabeled proteins will be visualized with a PhosphorImager. The same membranes then will be immunoblotted with either the antiSun1 serum or the antiV5 antibody. 5. Reconstitute Rebinant Protein Fragment Functionally into Detergent Micelles In order to prepare samples for NMR study, we will first choose suitable detergent micelles to reconstitute the rebinant protein fragment , use a direct dilution method to refold the proteins, and test the refolding of the target protein by radioligand binding. 6. NMR and Xray Crystallography Studies We will examine whether solution NMR studies of the micelle systems ( using different domains of the target protein ) will give suf
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