Isoelectric band

In Figure 3 is documented the resolution of a polyclonal antibody sanple exhibiting only a few closely spaced isoelectric bands. The antibodies were raised to the bacterial carbohydrates derived from Micrococcus lysodeiktikus in a rabbit from a colony outbred for simplicity of their clonotype patterns. After purification by affinity chromatography, the antibodies exhibited three major bands, with very close isoelectric points. To effectuate their separation, commercial Airpholine was subfractionated in the RIEF and a narrow cut, pH range 7.5 to 8.5, was used for the fractionation (8). This illustrates the resolution achievable in critical separations. 

For amino acids there is a band of about 2-3 pH units around the p/ where the net charge is zero. This is identified as the isoelectric band a pH value beyond this band on either side increases the amino acid solubility. For example, from equation (5.2.118a), we find that as Ch+ , increases, CAm+w increases therefore the amino acid solubility increases since CAm+w increases. Zumstein and Rousseau (1989) have illustrated that, for the amino acid L-isoleucine, the species dominates above pH = 2 leading to the... 

Because protein samples are actually ampholytes, when samples are loaded onto the gel and a current is appHed, the compounds migrate through the gel until they come to their isoelectric point where they reach a steady state. This technique measures an intrinsic physicochemical parameter of the protein, the pi, and therefore does not depend on the mode of sample appHcation. The highest sample load of any electrophoretic technique may be used, however, sample load affects the final position of a component band if the load is extremely high, ie, high enough to titrate the gradient ampholytes or distort the local electric field. 

Several electophoretic methods (SDS-PAGE, native PAGE and lEF) showed that fraction Q2 was almost pure, some faint contaminating protein bands were found (Fig. 9). The major band upon SDS-PAGE was at 42 kDa. lEF indicated an isoelectric point at pH 4.3 for the most prominent band... 

Separation of the purified PL1, PL2 and PL3 isoenzymes by SDS-PAGE yielded single protein bands that corresponded to a molecular mass of 42 kDa (6), 4 kDa higher than calculated from the deduced amino acid sequences. Isoelectric focusing revealed a pi of >10 for each isoenzyme similar to that of the basic Ech PLs (15). 

SDS-PAGE revealed only one protein band in the purified AE fractions with a MW of 42,000 D (Fig.2). Isoelectric focusing of AE showed that pi > 9. The amino acid composition of the purified AE is shown in table 1. 

However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3). 

The presence of the enzyme with lower pH optimum was not described yet [2, 11]. It gives only negligible peak by this pH (Fig. 2) in comparison with pH optimum by 5.0 (only 28 % from it) when pectate was used as a substrate. Isoelectric focusing (ruthenium red staining) showed poor and unclear bands noncorresponding the real activity of both enzymes (Fig. 5) in the pH area 4.5 -4.7. 

Evidence of pectic enzymes secreted by FORL species has been obtained (3). The total proteins of FORL subjected to isoelectric focusing were resolved into several bands widely distributed between pi 5.9 and 7.45. FORL (rj) presented one mayor band of activity with a pi of 7.0 however, FORL (r ) presented one characteristic and principal band at 7.45, slightly more basic than these reported by others authors (4). 

Zhu, Y. Lubman, D. M. Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations. Electrophoresis 2004, 25, 949-958. 

The column methods are much faster and are automated so that a much larger number of samples can be processed per unit time. An example of this technology, described in more detail in Chapter 10 by Lubman and coworkers, is shown in Figure 1.2, where the first dimension is from a chromatofocusing column, which gives separations in pI much like isoelectric focusing, only here the p/ axis is in bands instead of continuous pI increments. The second dimension is by reversed-phase liquid chromatography (RPLC). 

Two-dimensional electrophoresis is normally run so that proteins are separated from each other on the basis of a different molecular property in each dimension. The most commonly utilized method entails separation of proteins by isoelectric focusing (see below) in the first dimension, with separation in the second dimension being undertaken in the presence of SDS, thus promoting band separation on the basis of protein size. Modified electrophoresis equipment that renders two-dimensional electrophoretic separation routine is freely available. Application of biopharmaceuti-cal finished products to such systems allows rigorous analysis of purity. 

Two variations of the basic technique are isoelectric focusing and immuno-electrophoresis. The former offers improved resolution and sharper bands in the separation of weak acids, weak bases and ampholytes through the use of pH and density gradients superimposed along the potential gradient. The latter employs specific antigen-antibody interactions (Chapter 10) to visualize the separated components of serum samples. 

Most previous work on rat MUPs has been conducted with inbred or relatively inbred laboratory rat strains that are likely to exhibit considerably reduced phenotypic variation relative to the wild population, as we see in mice. As an initial exploration of MUP expression, we analysed urine from nine wild-caught male rats captured from several different populations in northern UK by isoelectric focusing electrophoresis (IEF). The protein banding pattern was very similar between individuals, consisting of two major and several minor bands. Peptide mass... 

In your notebook, make a drawing of the observed banding pattern, labeling both the hemoglobin and cytchrome C. Explain, in relevant detail, what you can determine about the isoelectric points of these proteins and what might happen in the gel if the buffer pH were changed from 7.9 to 3.9. 

As Smith (300) has shown by infrared spectroscopy, carboxylic acids are adsorbed either by hydrogen bonding of the carboxyl group or by proton transfer to the surface. Carboxylate absorptions were observed in the spectra. Very likely O " or OH ions acted as proton acceptors although no OH absorption bands could be detected after carboxylic acid adsorption. The isoelectric point of pure anatase is near pH 6.6 (305). 

As with SDS-PAGE gel methods, gel-based isoelectric focusing (lEF) methods have been used for decades to determine isoelectric point pi), which is an intrinsic property of protein molecules. Some complex proteins have multiple charge isoforms with multiple isoelectric points. These isoforms are separated as multiple bands in the lEF gel method. However, like other gel method, the lEF gel has limitations it is not automated, not reproducible, and not quantitative for pi determination. It is also labor intensive and requires large volumes of toxic reagents for staining. 

The pectate lyase activities secreted by Clostridium populeti, isolated from a poplar based methane digestor (28), were compared with those secreted by E. chrysanthemi and L. multiparus. Isoelectric focusing followed by overlay and activity analysis revealed the presence of one or two activity bands with pi values from 4.5 to 4.7 for both the C. populeti and the L. multiparus. The kinetic depolymerization profiles of C. populeti also revealed an exolytic/endolytic mechanism, typified by the high ratio of trimer and/or dimer to hexamer in the early stages of the reaction (Table I). 

Following the evaluation of isoelectric focusing, the results of unconcentrated CSF and sera, diluted to the corresponding concentrations, are classified into four categories by their banding patterns (S12) ... 

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