34监测蛋白质 Monitoring proteins B oEooE四 12 occongoL 0 255075100125150 0.5 1.5 C(mMy CMaci (mM) Figure 3.(A)Effects of KCl (A)and KGlu()on the MB/LDH binding. (B) Effect of MgCl2 on the binding. The concentrations of MB and ldh were 0. 4 and 1 uM. the buffer used was 20 mM Tris/ HCL, pH=7.8
3.4 监测蛋白质(Monitoring proteins)
34监测蛋白质 Monitoring proteins 0.8 0.6 04 D E A B1 B2 0.2 0 Figure 4. Effect of NAD on the binding of MB with LDH-5. The concentrations of MB and Ldh were 1 and 0.5 uM. the buffer used was 20 mM Tris/HCL, 0.05 mM MgCl2, pH=7.8
3.4 监测蛋白质(Monitoring proteins)
34监测蛋白质 Monitoring proteins a MB Nick Application of MBs in phosphorylation nucleotide a nucleotide B Hybridization and ligation studies a) real-time monitoring of nucleic acid ligation: Two oligonucleotides that are DNA Ligase complementary to opposite halves of Bindin the MB loop hybridize with the MB Ligation whereby a nick is formed, and the stem may be opened slightly. The dN ligase binds to the nick and catalyzes the ligation of the two short b) Oligo- oligonucleotides to form a longer nucleotide A PNK Phosphorylation oligonucleotide. The ligation product hybridizes with the MB to restore OH OH fluorescence HO b)Monitoring of nucleic acid Oligo- phosphorylation: Oligonucleotide ais nucleotide b Hybridization first phosphorylated at the 5-hydroxy group by the polynucleotide kinase OH MB The nick formed upon the hybridization A of oligonucleotide B and HO P DNA Ligase phosphorylated oligonucleotide Awith Ligation MB can be sealed by the dna ligase, whereupon the stem helix of the mb is opened, and fluorescence is restored
3.4 监测蛋白质(Monitoring proteins) Application of MBs in phosphorylation and ligation studies. a) Real-time monitoring of nucleic acid ligation: Two oligonucleotides that are complementary to opposite halves of the MB loop hybridize with the MB, whereby a nick is formed, and the stem may be opened slightly. The DNA ligase binds to the nick and catalyzes the ligation of the two short oligonucleotides to form a longer oligonucleotide. The ligation product hybridizes with the MB to restore fluorescence. b) Monitoring of nucleic acid phosphorylation: Oligonucleotide A is first phosphorylated at the 5’-hydroxy group by the polynucleotide kinase. The nick formed upon the hybridization of oligonucleotide B and phosphorylated oligonucleotide A with MB can be sealed by the DNA ligase, whereupon the stem helix of the MB is opened, and fluorescence is restored