Detailed Procedure

Permeabilize the cells with SLO using the protocol described in 

Replace the medium with solution LC with or without TeTx. For 96-microwells we add 50n.l solution while for 24-multiwells the 

TeTx treatment is terminated by removing the medium. The cells are then stimulated to secrete by adding LC or HC buffer for an additional 7 min period. 

At the end of the TeTx incubation it is possible to test the degree of cleavage of VAMPs. In the case of primary 3-cells and in INS-1 cells, where VAMPs can be detected in homogenates, the cells can be directly lysed in 300 il solubilization solution (SS) after removing the medium. We usually scrape the cells off using the pipette tip and transfer the lysate into an Eppendorf tube. Because of the presence of large amounts of DNA, the cell solution is very viscous. Therefore, the lysate is sonicated briefly (3x3 s) to disrupt the DNA, and is then boiled for 3 min. 

Clostridial Toxins and Endocrine Secretion Their Use in insulin-Secreting Cells 235 

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It would be difficult to over-estimate the extent to which the BET method has contributed to the development of those branches of physical chemistry such as heterogeneous catalysis, adsorption or particle size estimation, which involve finely divided or porous solids in all of these fields the BET surface area is a household phrase. But it is perhaps the very breadth of its scope which has led to a somewhat uncritical application of the method as a kind of infallible yardstick, and to a lack of appreciation of the nature of its basic assumptions or of the circumstances under which it may, or may not, be expected to yield a reliable result. This is particularly true of those solids which contain very fine pores and give rise to Langmuir-type isotherms, for the BET procedure may then give quite erroneous values for the surface area. If the pores are rather larger—tens to hundreds of Angstroms in width—the pore size distribution may be calculated from the adsorption isotherm of a vapour with the aid of the Kelvin equation, and within recent years a number of detailed procedures for carrying out the calculation have been put forward but all too often the limitations on the validity of the results, and the difficulty of interpretation in terms of the actual solid, tend to be insufficiently stressed or even entirely overlooked. And in the time-honoured method for the estimation of surface area from measurements of adsorption from solution, the complications introduced by... 

The hberated iodine is measured spectrometricaHy or titrated with Standard sodium thiosulfate solution (I2 +28203 — 2 1 VS Og following acidification with sulfuric acid buffers are sometimes employed. The method requires measurement of the total gas volume used in the procedure. The presence of other oxidants, such as H2O2 and NO, can interfere with the analysis. The analysis is also technique-sensitive, since it can be affected by a number of variables, including temperature, time, pH, iodide concentration, sampling techniques, etc (140). A detailed procedure is given in Reference 141. 

A double end point, acid—base titration can be used to determine both sodium hydrosulfide and sodium sulfide content. Standardized hydrochloric acid is the titrant thymolphthalein and bromophenol blue are the indicators. Other bases having ionization constants in the ranges of the indicators used interfere with the analysis. Sodium thiosulfate and sodium thiocarbonate interfere quantitatively with the accuracy of the results. Detailed procedures to analyze sodium sulfide, sodium hydro sulfide, and sodium tetrasulfide are available (1). 

Tables 16 and 17 Hst tke analytical test methods for different properties of interest. The Manufacturing Chemists Association, Inc. (MCA) has pubUshed the Chemical Safety Data Sheet SD 63, which describes in detail procedures for safe handling of use of toluene (46). The Interstate Commerce Commission classifies toluene as a flammable Hquid. Accordingly, it must be packaged in authorized containers, and shipping must comply with ICC regulations. Properties related to safe handling are autoignition temperature, 536°C explosive limits, 1.27—7.0 vol % in air and flash point 4.4°C, closed cup.

One standard test used to determine the presence of the coliform group is called the multiple-tube fermentation technique (sometimes called the presumptive test). If this test indicates the presence of these bacteria, then a confirmed test must be done. If only negative colonies or no colonies develop during this test, it is considered negative otherwise, a completed test must be undertaken. Positive results obtained in the completed test are evidence for the presence of coliform bacteria. Testing methods have been given by the APHA, and the detailed procedures contained therein should be consulted (20). 

A modified and detailed procedure is given for the preparation of the corresponding, and Co " xanthates ia good yields and high purity (18). A... 

The classical synthesis iavolves the dissolution of a 33% Sb—67% Zn alloy by hydrochloric acid the evolved gases contain up to 14% stibiae. A detailed procedure usiag a Sb—Mg alloy has also beea described (16). Aluminum hydride or alkaU metal borohydrides have been used to reduce antimony(III) ia acidic aqueous solutioa to produce stibiae. A 23.6% yield of stibiae, based oa the borohydride used, has beea reported (17). A 78% yield based oa Sb has beea obtaiaed by gradually adding a solutioa that is 0.4 Min SbCl and saturated ia NaQ, to aqueous NaBH at mol ratios of NaBH iSbQ. >10 (18). 

Detailed procedures, including computer programs for evaluating binary-interaction parameters from experimental data and then utihz-... 

We discuss below a high-impedance differential protection scheme to provide a detailed procedure to select PS Class CTs. 

The Environmental Protection Agency has detailed procedures for conducting air pathway analysis for Superfund applications (2). Decision network charts are given for all expected situations. 

Figure 13.3 Procedure for hot work on equipment that contains, or has contained, a flammable chemical (general summary only reference should be made to detailed procedures)...

All exterior hoods should be evaluated regularly. The evaluation procedures can be divided into detailed and simple procedures. Detailed procedures need special instruments and competence, whereas simple procedures may be performed daily. Since simple procedures do not directly measure the performance of the exhaust, it is usually necessary to calibrate them using detailed procedures. 

A detailed procedure for the use of MCPBA recently appeared in Reagents for Organic Synthesis by Fieser and Fieser. The commercially available MCPBA (Aldrich) is 85% pure the contaminant, m-chlorobenzoic acid, can be removed by washing with a phosphate buffer of pH 7.5. The epoxidation is usually performed as follows a solution of 3 -acetoxy-5a-androst-16-ene (2.06 g, 6.53 mmoles) in 25 ml of chloroform (or methylene dichloride) is chilled to 0° in a flask fitted with a condenser and drierite tube and treated with a solution of commercial MCPBA (1.74 g, 20% excess) in 25 ml chloroform precooled to the same temperature. The mixture is stirred and allowed to warm to room temperature. After 23 hr (or until TLC shows reaction is complete) the solution is diluted with 100 ml chloroform and washed in sequence with 100 ml of 10% sodium sulfite or sodium iodide followed by sodium thiosulfate, 200 ml of 1 M sodium bicarbonate and 200 ml water. The chloroform extract is dried (MgS04) and evaporated in vacuo to a volume of ca. 10 ml. Addition of methanol (10 ml) followed by cooling of the mixture to —10° yields 0.8 gof 16a,17a-epoxide mp 109.5-110°. Additional product can be obtained by concentration of the mother liquor (total yield 80-90%). 

BEST 9 Detailed procedures and checklists available Procedures developed using task analysis Integrated with training Noise levels at ideal levels Lighting design based on analysis of task requirements Temperature and humidity at ideal levels... 

Detailed procedures will only be required in unusual situations where the usual rules of thumb do not apply and the worker is likely to be in the knowledge-based mode. In Chapter 4, and case study 3 in Chapter 7, a systematic framework for developing procedures, in which their format and content is based on a detailed analysis of the tasks to be performed and the normal skill level of the person who will perform the tasks, will be described. 

If the whole task being analyzed has already been identified as being highly critical, then it may be necessary to subject every step to a PHEA. However, in most cases only those steps which have a high risk potential if errors occur will be examined in detail. Procedures for identifying critical tasks are described in Chapter 5. 

The top floor operators are usually highly skilled and experienced in many of the steps within the process. Therefore, a checklist may suffice as a job aid. However, new staff members may be sent to the top floor as part of their job-rotation scheme and a step-by-step procedure may be of benefit to them. Detailed procedures can be used either as job aids or as training aids. It is worthwhile, therefore, to develop both a checklist and a step-by-step procedure for the top floor job. 

Before discussing the kinds of kinetic information provided by potential energy surfaces we will briefly consider methods for calculating these surfaces, without going into detail, for theoretical calculations are outside the scope of this treatment. Detailed procedures are given by Eyring et ah There are three approaches to the problem. The most basic one is purely theoretical, in the sense that it uses only fundamental physical quantities, such as electronic charge. The next level is the semiempirical approach, which introduces experimental data into the calculations in a limited way. The third approach, the empirical one, makes extensive use of experimental results. 

A detailed procedure for the preparation of cholestanyl methyl ether from cholestanol has been published 11) and a survey of the usefulness of the reagent has also appeared 12). There appears to be no particular advantage to this procedure over the more convenient alternatives given above. 

Alcohols are the most frequently formed products of ester hydrogenolysis. The hydrogenation of esters to alcohols is a reversible reaction with alcohol formation favored at high pressure, ester at low pressure (/). Copper chromite is usually the catalyst of choice. Details for the preparation of this catalyst (/7) and a detailed procedure for hydrogenation of ethyl adipate to hexamethylene glycol (/[Pg.80]

Suction 6-inch pipe (using Cameron Tables—Table 2-22). To simplify calculations for greater accuracy, use detailed procedure of Chapter 2. 

Efficient and economical performance of distillation equipment is vital to many processes. Although the art and science of distillation has been practiced for many years, studies still continue to determine the best design procedures for multicomponent, azeotropic, batch, raul-tidraw, multifeed and other types. Some shortcut procedures are adequate for many systems, yet have limitations in others in fact the same might be said even for more detailed procedures. 

The periodic or critical load inspection in the field should be conducted by the crew with the inspector. For the periodic or critical load inspection, all foreign matter should be removed from surfaces inspected. Total field disassembly is generally not practical, and is not recommended, except as may be indicated in the detailed procedure for each tool. 

After pipework has been installed, it is essential that it be tested for soundness and purged, as required by Regulation 21 of the Gas Safety (Installation and Use) Regulations. Detailed procedures for these two operations are given in the British Gas publications Purging Procedures for Non-Domestic Gas Installations (lM/2) and Soundness Testing Procedures for Non-Domestic Gas Installations (lM/5). 

The only practical method for the preparation of anthra-quinone-a-sulfonates is that based upon the discovery 1 that in the presence of a small amount of mercuric salt anthraquinone is sulfonated chiefly in the a- rather than in the /3-position. Detailed procedures are described by Fierz-David,2 by Lauer,3 and by Groggins 4 the above directions are based largely upon the observations of Lauer.3... 

TnPht can be prepd by nitration of 2,4-din itrophenetole or by other methods given in Refs 1 10. Desvergnes (Ref 4) gives a detailed procedure for its lab prepn ... 

Detailed procedures for the synthesis ofa,o>-organofunctionally terminated siloxane oligomers with well defined structures have been given 50,66-67). Tables 6 and 7 provide the data on the synthesis and characteristics of aminopropyl and hydroxybutyl terminated polydimethylsiloxane oligomers prepared via anionic and cationic ringopening polymerization of octamethylcyclotetrasiloxane (D in the presence of appropriate disiloxanes, respectively. 

A detailed procedure is available on request from the submitters. 

Unfortunately, the two fuU papers on the silylation-amination of pyrimidine [49] and purine nucleosides [64] as discussed in Sections 4.2.3 and 4.2.4, were pubhshed in German and are thus not readily accessible, although a few detailed procedures from Sections 4.2.3 and 4.2.4 were subsequently published in English [65]. The third paper on the silylation-amination of aromatic hydroxy-N-hetero-cycles, however, as discussed in Section 4.2.5 was, fortunately, pubhshed in English [27]. 

The Fe-B nanocomposite was synthesized by the so-called pillaring technique using layered bentonite clay as the starting material. The detailed procedures were described in our previous study [4]. X-ray diffraction (XRD) analysis revealed that the Fe-B nanocomposite mainly consists of Fc203 (hematite) and Si02 (quartz). The bulk Fe concentration of the Fe-B nanocomposite measured by a JOEL X-ray Reflective Fluorescence spectrometer (Model JSX 3201Z) is 31.8%. The Fe surface atomic concentration of Fe-B nanocomposite determined by an X-ray photoelectron spectrometer (Model PHI5600) is 12.25 (at%). The BET specific surface area is 280 m /g. The particle size determined by a transmission electron microscope (JOEL 2010) is from 20 to 200 nm. 

The equation is balanced. Although we could have balanced this equation by inspection, the detailed procedure reveals its redox nature. 

Chapter 4 discusses the selection and optimization of mobile phases for successful separations in PLC. Chapter 5 details procedures for sample application and development of layers, and Chapter 6 complements Chapter 5 by dealing specifically with the use of horizontal chambers for the development of preparative layers, including linear, continuous, two-dimensional, gradient, circular, and anticircular modes. 

Catalysts Characterization Catalysts were characterized by nitrogen adsorption-desorption isotherms, XRD, XPS, TEM, and FT-IR. The concentration and the strength of the acid sites were determined using a combination of NHs-chemisorption and FTIR. Detailed procedures are given elsewhere [18, 19]. 

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