Buffer Recipes

If acetic acid and sodium hydroxide solution are used for preparation of the buffer, the amounts are calculated as follows  

6 = (moles NaOH) (moles CH3COOH - moles NaOH) 

1 mol/1 acetic acid wifh 0.061 mol/1 sodium hydroxide to get the expected buffer. 

If using acetic acid and sodium acetate, fhe following results are obtained  

6 = (moles Na acetate) (moles acetic acid) and 0.1 = (moles Na acetate) -1- (moles acetic acid). 

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Before robustness testing is started, precise specification of all method parameters is certainly needed (Table 6) else it may not be clear later which method has been validated. Possible settings for initial CE experiments are shown in Diagram 2. In particular, it is important to specify the buffer composition, e.g., by buffer recipes (Table 8). Otherwise, inadvertent variations in pH or ionic strength can lead to variability in selectivity. 

TABLE 8 Standard Buffer Recipes for Initial Experiments... 

The dependence of precision on different parameters has already been discussed. Precision is strongly dependent on the constancy of migration data. Thus, the stability of the EOF is most important. Buffer recipes describe clearly the preparation and avoid errors caused by, e.g., a poorly calibrated pH electrode. 

To successfully transfer CE methods, the use of buffer recipes and defined rinsing procedures is strongly recommended. 

NOTE The designation m stands for molality. Masses in die buffer recipes below are apparent masses measured in air. 

The limited volume of the total cellular material from biopsy samples makes it necessary to use a slight modification of the lysis buffer recipe as compared with the cell culture lysis buffer. The following protocol describes a method for preparing whole cell lysates from LCM cells. The limited biopsy sample generally precludes the use of total protein assays before microarray printing. To compensate for this, one microarray slide is stained for total protein using a Sypro Ruby Protein blot stain (see Subheading 3.3.3.). 

Two basic gel running buffer recipes are standard, Tris-acetate (TAE) and Tris-borate (TBE) we use the latter, and this is included in the recipes given below. 

The pH of the buffer solution in which the analysis takes place must also be controlled. Most enzymes have a pH dependence of their activity of the type shown in Figure 4 B, and pH should be controlled to within 0.02. The optimum pH, and the range over which the enzyme is active, vary widely from one enzyme to another. These phenomena arise from the effects of pH on the structure of the enzyme itself, on the affinity constant between the enzyme and the substrate, on V ax. and also on any coupled indicator reaction that is used. The choice of buffer recipe for any given pH may be important. The ionic strength of the buffer and the salts contained in it can influence the rate and mechanism of the main and coupled reactions, sometimes with unpredictable results. It may be necessary to choose a buffer whose properties represent a compromise between the ideal conditions for the main reaction, and the ideal conditions for the indicator leaction(s). Immobilized enzymes (Section 9.1.7) often have pH (and temperature) dependence significantly different from their soluble counterparts. This all points to the value of establishing and maintaining a well-defined buffer system for enzymatic analyses. 

Tris-glycine (pH 8.5) was used as the running buffer for Hk, Mpi, and Pgi. Tris-malate (pH 7.8) was used as the running buffer for 6Pgd (see Richardson et al. (1986) for buffer recipes). Enzyme stains were modified from Hebert and Beaton (1989). Numerous side-by-side comparisons of electromorphs were made to confirm relative electrophoretic mobilities alleles were assigned letter codes with A representing the... 

Buffers are frequently added to emulsion recipes and serve two main purposes. The rate of hydrolysis of vinyl acetate and some comonomers is pH-sensitive. Hydrolysis of monomer produces acetic acid, which can affect the initiator, and acetaldehyde which as a chain-transfer agent may lower the molecular weight of the polymer undesirably. The rates of decomposition of some initiators are affected by pH and the buffer is added to stabilize those rates, since decomposition of the initiator frequently changes the pH in an unbuffered system. Vinyl acetate emulsion polymerization recipes are usually buffered to pH 4—5, eg, with phosphate or acetate, but buffering at neutral pH with bicarbonate also gives excellent results. The pH of most commercially available emulsions is 4—6. 

Prepare liposomes containing PE by any desired method. For instance, the common recipe mentioned in Section 1 (this chapter) that involves mixing PC cholesterol PG PE in a molar ratio of 8 10 1 1 may be used. Thoroughly emulsify the liposome construction to obtain a good population of SUVs. The final liposome suspension should be in 20mM sodium phosphate, 0.15M NaCl, pH 7.2. Adjust the concentration to about 5 mg lipid/ml buffer. 

In this method the sample is acidified and the inorganic carbon is removed with nitrogen. An aliquot is resampled for analyses. Buffered persulfate is added and the sample is irradiated in the ultraviolet destructor for about 9 min. The hydroxylamine is added and the sample stream passes into the dialysis system. The carbon dioxide generated diffuses through the gas-permeable silicon membrane. A weakly buffered phenolphthalein indicator solution is used as the recipent stream, and the colour intensity of this solution decreases proportionately to the change in pH caused by the absorbed carbon dioxide... 

TABLE 5.2 Recipes for Some of the More Popular Buffer Solutions... 

For example, to prepare a pH = 9 buffer solution, one would prepare a solution of ammonium chloride (refer to Table 5.1), and then add a solution of sodium hydroxide while stirring and monitoring the pH with a pH meter. The preparation is complete when the pH reaches 9. The required conjugate acid-base pair would be NH3 - NHj. Recipes for standard buffer solutions can be useful. Table 5.2 gives specific directions for preparing some popular buffer solutions. 

In Experiment 14, a recipe for a pH =10 buffer solution is given. This recipe calls for dissolving 35 g of NH4C1 and 285 mL of concentrated ammonium hydroxide (15 M) in water in the same container and diluting with water to 500 mL. Calculate the pH of the buffer using the data given, and confirm that it really is 10. The Ka for the ammonium ion is 5.70 x 10. ... 

Buffer pH, molarity, recipe weight or volume of all chemicals used... 

Three preliminary experiments were carried out the first done as described above, the second utilized a modified buffer containing protectants, and the third utilized both the modified buffer and a hemoglobin gel step to remove proteases. The modified buffer had the following recipe 50 mM EPPS-KOH buffer at pH 8.6, 20% (v/v) glycerol, 1.0 mM EDTA, 1.0 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM leupeptin, and 0.05% / -mercaptoethanol. 

For 96 samples, prepare 100 samples premix. Assemble the following recipe Donor vector (pDONR221 150 ng/pl) 50 pi, BP reaction buffer (5x) 100 pi, TE buffer 150 pi, BP Clonase enzyme mix 100 pi. 

Typical emulsion polymerization recipes involve a large variety of ingredients. Therefore, the possibilities of variations are many. Among the variables to be considered are the nature of the monomer or monomers, the nature and concentration of surfactants, the nature of the initiating system, protective colloids and other stabilizing systems, cosolvents, chain-tranfer agents, buffer systems, short stops, and other additives for the modification of latex properties to achieve the desired end properties of the product. 

Lists of reagents, and recipes for working solutions and reagents are provided in Tables 1.4.1 and 1.4.2, respectively. The lactate reagent is prepared as per the manufacturer s instructions. The reagent required for the deproteinisation step is a solution of perchloric acid 1 mol/1 (dilute 8.6 ml of HCL04 70% in 100 ml distilled water or 11 ml of HCL04 60% in 100 ml distilled water). That required for the neutralisation is phosphate tripotassic buffer 1 mol/1 add 5.3 g of phosphate tripotassic in 25 ml distilled water. This solution will be stable for 1 year at 25°C. 

Emulsion Polymerization. Poly(vinyl acetate)-based emulsion polymers are produced by the polymerization of an emulsified monomer through free-radicals generated by an initiator system. An emulsion recipe, in general. contains monomer, water, protective colloid or surfactant, initiator, buffer, and perhaps a molecular weight regulator. 

The initiators used m vinyl acetate polymerizations are the familiar free-radical types, Buffers are frequently added to emulsion recipes. Vinyl acetate emulsion polymerization recipes are usually buffered to pH 4-5. The pH of most commercially available emulsions is 4-6. 

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