Buffer composition

TABLE 4.4 Effect of Buffer Composition on Mass Recovery of Proteins from TSK-GEL G3000SW< ... 

Freeze-drying of aequorin. The process of freeze-drying always results in some loss in the luminescence activity of aequorin. Therefore, aequorin should not be dried if a fully active aequorin is required. The loss is usually 10% or more. The loss can be somewhat lessened by adjusting the buffer composition the use of 100 mM KCl and some sugar (50-100 mM) seems to be beneficial. The buffer composition used at the author s laboratory is as follows 100 mM KCl, 50 mM glucose, 3 mM HEPES, 3 mM Bis-Tris, and at least 0.05 mM EDTA, pH 7.0. 

A computer algorithm SEDNTERP [33,34] has been developed for facilitating the correction in Eq. 3. There is no longer any need (except for imusual solvents) to look up solvent densities and viscosities in the Chemical Rubber Handbook or other data books—a user of the algorithm just has to specify the buffer composition and the temperature of the measurement and the correction is done automatically. 

Further complication is apparent when the reaction is investigated in HzPOJ-HPO buffers while the rate shows a first-order dependence on total [Cr(Vl)] and [As(lll)], a complex dependence on the buffer composition is found, indicating two activated complexes of composition HzPOX HCrO -As(III) and HP04 HCr0J-As(III), which correspond to attack of HCrPO upon neutral As(III) and As(0H)20" respectively. 

This novel enzyme was the only esterase able to release acetyl from sugar beet pectin and removed about 30% of the total acetyl groups present. It also caused the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, in small amounts. PAE had an apparent molecular weight of 60 kDa and showed optimal activity at pH 5.5 and a temperature of 50 C. The enzyme is sensitive to buffer composition and requires a bivalent cation for optimal activity and stability. In purified form this enzyme proved unstable, especially in phosphate buffers. 

Fig. 1.9 Dependence of pH on the buffer composition according to the Henderson-Hasselbalch equation (1) acidic buffer (Eq. 1.4.26) (2) basic buffer. Calculation for K A = K B = 10-4, s = 0.1 mol dm-3...

The following protocol should be compared to the method described for SATA thiolation in Chapter 1, Section 4.1. Although the procedures are slightly dissimilar, the differences indicate the flexibility inherent in the chemistry. For convenience, the buffer composition indicated here was chosen to be consistent throughout this section on enzyme-antibody conjugation using SMCC. Other buffers and alternate protocols can be found in the literature. 

The kidney exerts control over the concentration of bicarbonate. The initial filtrate of the kidney has the same buffer composition (except for the proteins) as serum. Based on the pH of the initial filtrate, the kidney can decide to reclaim the initially filtered bicarbonate and place it back in the serum, or it can decide to just let it go out of the body. If the kidney does not reclaim bicarbonate, the serum bicarbonate concentration falls. If the kidney reclaims more bicarbonate than normal, the bicarbonate concentration rises. 

This procedure can be repeated for other coating conditions (time, temperature, pH, buffer compositions, blocking steps, wash buffers, etc.). 

In general terms, the variation from laboratory to laboratory (between-laboratory) was greater than that attributed to the analytical error displayed within laboratories (intralaboratory). There are many reasons for the interlaboratory variation that can be attributed to operational parameters such as mobile phase flowrate, mobile phase and buffer composition, vaporiser temperature, tip temperature and source temperature. 

In some cases, sample preparation for CZE requires only the dilution of the sample, mostly to accommodate detection (for signal and linearity of response). However, as was previously mentioned, sample characteristics such as viscosity, buffer composition (pH and excipients), and salt content can especially affect electrophoretic injection and performance. 

Manipulation of buffer pH or use of additives can easily vary separation selectivity. In contrast to gel electrophoresis, a single capillary can be used to evaluate the effect of buffer composition or pH on resolution. In CZE the capillary is simply filled with a fresh electrolyte of the chosen composition between analyses. 

Dunn SD. 1986. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem 157 144-153. 

Effects of buffer composition on electroosmotic flow in capillary electrophoresis. /. Microcol. Sep. 2, 176-180. 

Running buffer composition, including a detailed preparation procedure with the order of addition of the components... 

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. 

We will first review the principles of indirect detection, how it works, and what are its requirements. Thereafter, we will focus on considerations concerning buffer composition and sample preparation. The goal is to develop tools that can be used today with commercially available instruments present in many pharmaceutical companies. We will also review available publications and, in an addendum, describe a complete assay for phosphate as a counterion and an impurity. 

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