Ampholytes carrier

Ampholytes represent complex mixtures of homologous polyamino polycarboxylic or polysulfonic acids with close pi and pK values [135,136]. The differences in pi values represent inter-species differences, while the differences in pA values are related to a single species (intra-species differences). Usually the following ranges of pH are covered 3.5-10.0, 2.0-4.0, 3.5-5.0, 4.0-6.0, 5.0-7.0, 5.0-8.0, 6.0-8.0, 

When studied by gel permeation chromatography with respect to the distribution of their molecular weight, it was shown that most of the ampholyte components exhibit a relative molecular mass of between 300-1000, with few species found at a value of 5000 [140]. This is valuable information as long as it might be necessary to 

IEF was first successfully applied to proteins in 1938, when it was used to separate the protein hormones vasopressin and oxytocin from tissue extracts. Twenty years later, ampholytes were first focused in a continuous pH gradient, stabilized by a dense sucrose medium, as an alternative to the multicompartment method. The continuous pH gradient in the sucrose medium was established by allowing acid and base to diffuse into opposite ends of the sucrose medium, held in a U-cell, from their respective electrode chambers. The stabilization of this continuous pH gradient with carrier ampholyte species led to modern IEF methods. 

The first studies of carrier ampholytes were conducted using amino acids and dipeptides, but these species did not work well because their pK values for the amino and carboxylate groups are too far removed from their pi values. After they were prefocused, these species had very low buffering capacity. Good 

Carrier ampholyte-based IEF methods are commonly used in situations where very high resolution of proteins according to their pi values is not required. Several problems exist with the use of carrier ampholytes that limit their resolving power. These include the low and uneven ionic strength that results in smearing of the most abundant proteins in the sample, the uneven buffering capacity and conductivity, the unknown chemical environment, a low sample loading capacity, and a 

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Shimao, K, Mathematical Simulation of Steady State Isoelectric Eocusing of Proteins using Carrier Ampholytes, Electrophoresis 8, 14, 1987. 

Rabilloud, T., Valette, C., and Lawrence, J. J. (1994). Two-dimensional electrophoresis of basic proteins with equilibrium isoelectric focusing in carrier ampholyte-pH gradients. Electrophoresis 15, 1552-1558. 

With carrier ampholytes, concentrations of about 2% (w/v) are best. Ampholyte concentrations below 1% (w/v) often result in unstable pH gradients. At concentrations above 3% (w/v), ampholytes are difficult to remove from gels and can interfere with protein staining. 

A fundamental problem with IEF is that some proteins tend to precipitate at their pi values. Carrier ampholytes sometimes help overcome pi precipitation, and they... 

IPGs into mirrors. An easy way to stain IPGs is to immerse them for 1 h in colloidal CBB G-250 followed by two 10-min water washes. There is also a version of SYPRO Ruby stain specifically formulated for use with both carrier ampholyte and IPG-IEF gels. 

IEF is similar in concept to conventional gel IEF a stable pH gradient is formed in the capillary using carrier ampholytes, and proteins are focused in the gradient at their pis. The major difference in performing IEF in the capillary format rather than slab gel is the requirement for mobilizing focused protein zones past the detection point. IEF is described in Capillary Isoelectronic Focusing. ... 

As previously mentioned, resolution in CIEF strongly depends on the ampholyte composition. The estimated maximum resolving power of IEF is 0.02 pH units when carrier ampholytes are used to create the pH gradient.99 The Law of Monotony" formulated by Svensson in 1967 states that a natural pH gradient increases continually and monotonically from the anode to the cathode that the steady state does not allow for reversal of pH at any position along the gradient and that two ampholytes (in stationary electrolysis) cannot be completely separated from each other unless the system contains a third ampholyte of intermediate pH (or pi). The latter explains why better resolution is obtained when mixing ampholytes from different vendors and production batches as the number of ampholytes species increases, the chance that one or more ampholytes have intermediate pi relative to those of the sample components also increases. 

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. 

Wallevik, K. 1973. Isoelectric focusing of bovine serum albumin. Influence of binding of carrier ampholyte. Biochim. Biophys. Acta 322, 75-87. 

Dissolve 5.04 g urea (final 8 M) and 788 (J.1 carrier ampholyte mixture (e.g., Pharmalyte Amersham Pharmacia Biotech final 7.5%) in water to 10.5 ml. Add a reducing agent (60 mM dithiothreitol) and/or a detergent (0.5% [w/v] Triton X-100, CHAPS, or octyl glucoside), if desired. Make fresh. 

Govi, M., Bonoretti, G., Ciavata, C., and Sequi, P. (1994). Characterization of soil organic matter using isoelectric focusing a comparison of six commercial carrier ampholytes. Soil Sci. 157, 91-96. 

Shang TQ, Ginter JM, Johnston MV, Larsen BS, McEwen CN. Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures. Electrophoresis 2003 24 2359-2368. 

The pH gradient in cIEF is produced by the use of reagents, known as carrier ampholytes, that are zwitterionic and are chosen so that the... 

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