In protocols

Although the previous protocol suggests it is not necessary to deprotonate the sulfonamide prior to exposure to the zinc carbenoid, a experimentally simpler procedure can be envisioned wherein the alcohol and promoter are deprotonated in a single flask (Fig. 3.15). In protocol IV, the alcohol and promoter are combined in flask A and are treated with diethylzinc, thus forming the zinc alkoxide and zinc sulfonamide. In sub-protocol IVa, this solution is transferred to flask C which contains the zinc carbenoid. Sub-protocol IVb represents the reversed addition order. Sub-protocol IVa is not only found to be the superior protocol in this sub-set, it is found to out-perform all of the previous protocols Despite the persistence of the induction period, a large rate enhancement over the uncatalyzed process is observed. This considerable rate enhancement also translates to a reduction in the overall reaction time when compared to sub-protocols la and Ilia. Selectivity rises... 

PROTOCOL AMENDMENT(S) n NEW PROTOCOL n CHANGE IN PROTOCOL n NEW INVESTIGATOR... 

Application rates in protocols can be expressed in various ways, and the field staff should be aware of these types of expressions and understand their meanings, e.g.,... 

Representative data for [ H]acetylcholine binding to the membrane-bound Torpedo nAChR. Bindng was measured either by equilibrium dialysis (closed circles) as described in Protocol 4.1 or by centrifugation (open squares, see Protocol 4.2). Estimated Kd values from nonlinear regression curve fitting were 12 nM and 10 nM, respectively with corresponding Rq values of 0.14 ulM and 0.135 ulM... 

Plasma non-esterified fatty acids (NEFA) were elevated by infusion of a lipid emulsion and heparin with a constant rate of 1.5 ml/min (lipid emulsion, protocols [1] and [3]) and 0.4 lU/kg per minute (heparin). In protocol [2] a solution of 0.9% saline was infused as a control for the lipid emulsion. 

Increase of IMCL content was also clearly less pronounced under lipid infusion without hyperinsulinemic conditions (protocol [3]). This protocol was carried out in a very similar way by Krssak et who reported that elevation of FFA without hyperinsulinemia does not alter IMCL content. In contrast to those findings are the results of Boden et al. who observed a massive increase of IMCL under similar conditions as in protocol [3], but only a low increase after protocol [1]. The reasons for these discrepancies are still debated. 

It is therefore essential to incorporate procedures that will ensure not only that the clinical data are collected in a uniform and similar manner at each investigating centre but that they are also handled and analysed in an identical manner. Measures such as the use of compatible validated computer systems and similar databases will allow the merging of data. At each centre, critical efficacy data should be determined with identical procedures and, where appropriate, these should be specified in protocols... 

Instructions for handling study drug - may be in protocol or investigator s brochure... 

Use a pen with permanent ink, and NOT a pencil, to make your entries. Date each entry, and be sure to put a date next to comments that you find necessary to add on a later occasion. (Such careful chronicling will allow you to recollect why you made certain changes in protocol or observations, especially those based on some later realization.)... 

If a methionine auxotroph is not used, the methionine within the culture can be replaced by SeMet using a methionine biosynthesis inhibition or poisoning method such as that outlined in Protocol 2.7. This method has the advantage of using any E. coli strain available and thus the initial growth of the culture is not reduced, however the method is more laborious than using the methionine auxotroph. 

Mass spectrometry (MS) is widely used to ascertain the purity, total mass of the protein produced, and detect any covalent modifications (Cohen and Chait, 2001). Both electrospray ionization (ESI) and MALDI may be used although for intact proteins ESI has the advantage of being accurate to 1 Da. Using the simple protocol described in Protocol 2.11, the MS of whole protein samples can be readily automated without the need for sample preparation. This method has proved successful for the... 

Once a hit has been found, the next step is to conduct a finer screen around these conditions by varying the concentrations of protein, precipitant, addition of additives, etc. In most cases, more than three different ingredients may be needed in each trial, making it difficult to manually pipette all the ingredients into one drop directly under the oil. Thus the procedure in Protocol 3.2 should be followed. 

The protocol for setting up microbatch experiments containing membrane proteins is identical to that described in Protocols 3.1 and 3.2. 

When mixed, draw a drop of a volume of your choice with a pipette tip and dispense under the oil as described in Protocol 3.1. 

While the first point was addressed directly in Protocol 7.1, we will address the other points successively in the next paragraphs. 

In Protocol 7.3 we show a typical command file for the AMoRe package (Navaza, 2001), one of the most widely used packages for MR. 

Use the dry residue for digestion and phosphate determination as described in Protocol 1.3.2. 

The quantitative determinations of the monosaccharides ribose and deoxyribose are given in Protocols 1.2.2 and 1.2.3, respectively. The following protocol is useful for all monosaccharides. 

Electrode buffer is Soln. D, the sample buffer is prepared by addition of a trace bromophenol blue and sucrose to Soln. D. Electrophoretic conditions are similar to that given in Protocol 2.1.1. 

This protocol is adapted for analysis of proteins with pi > 8. The other deviations from SDS-PAGE are described in Protocol 2.1.8. 

Identify the ligate by specific antibodies as described in Protocol 2.5.4 (Western blot). 

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