Automation protocols

The most notable effort in standardizing plant automation protocols... 

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

PfefFer, de Vries and coworkers developed the use of ruthenacycles, based on chiral aromatic amines as enantioselective transfer hydrogenation catalysts. These authors were able to develop an automated protocol to produce these catalysts by reacting ligand and metal precursor in the presence of base, KPFS in CH3CN. After removal of the solvent, isopropanol was added followed by the substrate, acetophenone, and KOtBu. In this way, a library of eight chiral... 

The need for automated protocols is apparent from the strategy adopted by AMoRe to circumvent the problem that the score of the rotation function (RF) is far from being perfect and does not always rank the solutions correctly (Navaza, 2001). Indeed, it is often observed that the true solution is not the top solution, with many false positives. Hence, AMoRe runs a translation function (TF) for each of, typically, the top 50 or 100 solutions of the rotation function. This is actually quite rapid as TF is based on FFT then, the first 10 solutions of each of these TF runs is in turn refined using a very effective implementation of rigid-body refinement (Navaza, 2001). 

It seems pretty clear that easy-to-use and web-interfaced, automated protocols will be more and more useful in the near future. The possibility of combining different tools at different steps of the integrated process makes it inevitable that more of them will be developed and open to the pubhc. What is good about this kind of approach is that... 

DNA sequences can be determined and DNA polymers synthesized with simple, automated protocols involving chemical and enzymatic methods. 

In this section, we present the development of an automated protocol for prostate tissue histology [164] from infrared spectroscopic imaging data as an example of the techniques described (Fig. 8.11). The data is three dimensional with x-y—axes representing the image plane and the 2-axis representing the spectral dimension. After data acquisition, two important pre-processing steps, namely baseline correction and de-noising, are performed. Since the entire data set is derived from human tissue samples, the spectra have similar characteristics and, therefore, a manually chosen set of pre-defined wave number could be used as the reference points for baseline correction. It is... 

Given our success with the use of trichloroacetmidate donors, we sought to explore the use of glycosyl phosphates in an automated protocol.1... 

A batch of resin-bound 6.48 was prepared in a reaction flask and divided into 50-mg portions in each well of the 96-weU reaction block of the synthesizer. The synthesis of the library on the 96-well reaction block required around 8 h of instrument time and could have easily been repeated with different monomers in order to enrich the chemical diversity. Fully automated protocols provide access to 24-hr/day, seven-days/week operation and can thus maximize the use of an automated SP synthesizer the same protocol has been subsequently adapted to a higher throughput SP synthesizer (164) and has provided a 3072-member discrete SP library of tricyclic compounds (165). 

Figure 8.15 Software automated protocols for the synthesis of the solution-phase, discrete triazine library L3.

Automation must not affect the robustness of the assay, because automated protocols cannot afford last-minute adjustments or modifications. 

One of the critical steps in qualitative and quantitative analysis is the sample preparation procedure. Sample preparation step can affect specificity, sensitivity, accuracy, precision, and throughput of a bioanalytical procedure. In addition to development and optimization of the chemistry involved in sample processing, the use of semiautomated or fully automated protocols has been... 

An automated protocol for sequentially analysing MQ spectra to obtain reasonable estimates of spectral parameters then analysing IQ spectra has been developed.61 The analysis of MQNMR spectra has frequently been used to provide good initial estimates of parameters to permit the analysis of IQ spectra where these are well resolved. 

This LC-MS-based methodology can be automated to fit specialized needs within drug discovery based on throughput [62], The recent application of mass defect filtering [90] and high-resolution accurate mass analysis [33] provides further automated protocols for metabolite identification (see Chapters 5 and 6 for more on this topic). 

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