Lab Report On The Concentration Of Compound 1 For 30 Min At Room Temperature

1489 Words Dec 28th, 2016 6 Pages
for 30 min at room temperature. Then, trypsin/chymotrypsin- digested samples of KPNB1 obtained from the UV-crosslinking experiments with various concentrations of compound 1 were analyzed by nanoflow liquid chromatography and tandem mass spectrometry. The tandem mass spectra of the trypsin/chymotrypsin-digested KPNB1-compound 1 adducts showed 10 peptides coupled to compound 1 (Table S1, the Supplementary Information). Among these adducts, we found that the amounts of three of the products, 256MG*PALF261, 475SS*LAEAAY482, and 483EAADVADDQ*EEPATY497 (amino acid numbering taken from IMB1_HUMAN, SwissProt: Q14974), increased in a dose-dependent manner, suggesting that compound 1 binds at the specific site of KPNB1 (Table 1). Even though relative intensities of these peptides increased up to 10 μM compound 1, the amounts of these peptides markedly decreased at higher concentrations of compound 1 (100 μM), probably owing to the non-specific binding of compound 1 to KPNB1, which would have interfered with specific binding.
Table 1. Changes in the relative intensities of peptides with compound 1 at various concentrations

Because the results of the UV-crosslinking experiment showed the three addition sites of compound 1 to KPNB1, a Job’s plot analysis was constructed to determine the binding stoichiometry of the KPNB1-compound 2 complex. In this approach, the molar fraction of KPNB1 and compound 2 was varied, while keeping the total concentration ([KPNB1]+[2]) constant. The…

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