Ampholytes buffer

Figure 1. Fractionation of proteins in the culture filtrate of Trichoderma reesei according to their pi values Xyl, xylanase Ara, arabinosidase AE, acetyl esterase / X, /3-xylosidase aG, a-glucuronidase / G, / -glucosidase CBH, cellobiohydrolase EG, endoglucanase. Chromatofocusing was performed in a PBE-94 anion exchange resin (Pharmacia) with a pH-gradient created by ampholyte buffers (Pharmacia). Solid line, A dotted line, pH. (Reproduced with permission from ref. 24. Copyright 1988.)...

At or in the proximity of their pi values proteins exhibit a minimum total charge and reduced solubility in the electrolyte solution [350,370,385]. This increases the probability of aggregation, and is further enhanced by the low ionic strength of the ampholyte buffer. Under these conditions protein precipitation results from hydrophobic interactions. These interactions can be suppressed by addition of additives such as ethylene glycol (10-40 %), non-ionic or zwitterionic surfactants (1-4 %), or sorbitol to the ampholyte buffer. 

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 nun) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 pg of protein in 10 pi of 1 % glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HCl buffer at pH 8.0 with CaClj M.- Broad pi Calibration Kit protein (Pharmacia), samples of 5 pg of protein were applied. 1.-Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Shiau et al. [73] directly measured the microclimate pH, pHm, to be 5.2-6.7 in different sections of the intestine (very reproducible values in a given segment) covered with the normal mucus layer, as the luminal (bulk) pH, pH/, was maintained at 7.2. Good controls ruled out pH electrode artifacts. With the mucus layer washed off, pHm rose from 5.4 to 7.2. Values of pHfo as low as 3 and as high as 10 remarkably did not affect values of pHm. Glucose did not affect pHm when the microclimate was established. However, when the mucus layer had been washed off and pHm was allowed to rise to pHfo, the addition of 28 mM glucose caused the original low pHm to be reestablished after 5 min. Shiau et al. [73] hypothesized that the mucus layer was an ampholyte (of considerable pH buffer capacity) that created the pH acid microclimate. 

Figure 9.5 Generation of a pH gradient by ampholytes within a capillary flanked by an acid as anodic solution and base as cathodic solution. Ampholyte solutions are composed of high numbers of low-molecular weight amphoteric electrolytes (from which the name is derived) with slightly different pi values. Because ampholytes possess buffering capacity, they maintain a pH value in the specific area occupied by the different molecular species. The sample, which is also amphoteric, focuses in between ampholytes with higher and lower pi. To achieve resolution, there must be at least one ampholyte with a pi intermediate to the two sample components of interest.

Sample buffer. 8 M urea, 2 M thiourea, 4% w/v 3-[(3-chola-midopropyl)dimethylammonio]propanesulfonic acid (CHAPS), 20 mM dithiothreitol (DTT), 0.5%v/v carrier ampholyte. 

An IEF/CGE separation for proteins has been achieved on a PDMS chip. Microfluidic valves were used to prevent intermixing between the two separation buffers used in IEF and CGE separations. The IEF ampholyte was very sensitive to high buffer concentration, but a small amount of ampholyte in the CGE did not affect its separation resolution [449]. 

---