We present a disposable microarray hybridization chamber with an integrated micropump to speed up diffusion based reaction kinetics by generating convective flow. The time-to-result for the hybridization reaction was reduced from 60 min (standard protocol) down to 15 min for a commercially available microarray. The integrated displacement micropump is pneumatically actuated. It includes two active microvalves and is designed for low-cost, high volume manufacturing. The setup is made out of two microstructured polymer parts realized in polycarbonate (PC) separated by a 25 μm thermoplastic elastomer (TPE) membrane. Pump rate can be controlled between 0.3 μl s(-1) and 5.7 μl s(-1) at actuation frequencies between 0.2 Hz and 8.0 Hz, respectively.
Print) 1362-3028 (Online) Journal homepage: http://www.tandfonline.com/loi/tmph20 31 P and 13 C chemical shielding tensors in the phosphoenolpyruvate moiety from rotary resonance recoupling 13 C and 31 P MAS and single crystal 31 A 31P and I3C NMR study of powder and single crystal samples of two phosphoenolpyruvate (PEP) compounds, the tris-ammonium salt monohydrate NH4)3(PEP).H20 (l), and the mono-ammonium-salt (NH4)(H,PEP) (2) is presented. The A P chemical shielding tensors in 1 are measured by 31P single crystal NMR on four minuscule samples and assigned without ambiguity by exploiting the orientation-dependent 31P-31P dipolar splittings of the resonance lines. The orientation of the 31P chemical shielding tensor is discussed in terms of the C2"-and C3-type distortions of the phosphate P04-coordination sphere. From I3C MAS NMR experiments with 31P rotary resonance recoupling on polycrystalline powder samples the orientations of the 31P chemical shielding tensors in l and 2 are obtained, for l in very good agreement with the 31P single crystal NMR results. Only some of the orientational parameters of the three 13C chemical shielding tensors in the PEP moiety of 1 could be derived from 13C MAS NMR experiments with 31P rotary resonance recoupling.
Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPγS are widely used to stabilize rapidly hydrolyzing protein‐nucleotide complexes and to investigate biochemical reaction pathways.Here we describe the chemical synthesis of guanosine 5′‐O‐(γ‐amidotriphosphate) (GTPγNH2) and a new synthesis of guanosine 5′‐O‐(γ‐fluorotriphosphate) (GTPγF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on 31P, 19F and 1H NMR resonances are tabulated. For GTPγNH2 the enthalpy of magnesium coordination is ΔH° = 3.9 kcal·mol−1 and the association constant Ka is 0.82 mm−1. The activation energy for GTPγNH2·Mg2+ complex formation is ΔH‡ = 7.8 ± 0.15 kcal·mol−1, similar to that for the natural substrate GTP. For GTPγF we obtained a similar enthalpy of ΔH° = 3.9 kcal·mol−1 while the magnesium association constant is only Ka = 0.2 mm−1. The application of both guanine nucleotide analogues to theGTP‐binding protein Ras was investigated. The rate of hydrolysis of GTPγNH2 bound to Ras protein lay between the rates found for Ras‐bound GTPγS and GppNHp, while Ras‐catalysed hydrolysis of GTPγF was almost as fast as for GTP. The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies.
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