Antibodies isoelectric point

Capillary isoelectric focusing (CIEE), Isoelectric point (p7) Proteins and peptides, glycoproteins, monoclonal antibodies, isoelectric point determination, peptide mapping... 

CV measurements showed that the reversible eleetrode reaetion of the [Fe(CN)6]" redox eouple was suppressed to some extent by the treatment with the DNA. The addition of the anti-DNA antibody further suppressed the redox reaetion thus decreasing the magnitudes of the CV peak currents. This is most likely caused by a steric hindrance of the bulky protein, which binds to the DNA double strands on the electrode surface, to mainly reduce the effective area of the electrode. The electrostatic repulsive effect may also contribute to the electrode response, since the isoelectric point of mouse IgM is commonly in the range of 4.5 to 7.0. Figure 11 shows the relationship between the decrease in the anodic peak current (A/p ) and the antibody concentration. As seen in this figure, the electrode system responded to the anti-DNA antibody in the concentration range of 1 — 100 nM. For the case of the mouse IgM, which does not interact with double-stranded DNA, the present system gave almost no response. The sensor did not respond to other serum proteins as well (data not shown). 

Difficulties in detecting nucleolin on the cell surface could be explained by its very low concentration in this compartment (Hovanessian et al, 2000). Moreover, this cell surface expressed nucleolin protein has a different isoelectric point and it is recognized by only one monoclonal antibody (mAb D3) in its native conformation (Hovanessian et al, 2000). This probably reflects specific post-translational modifications undergone by nucleolin on the cell surface. Consistent with this hypothesis, extracellular nucleolin is a substrate of ecto-protein kinases including casein kinase II (Dumler et al, 1999 Jordan et al., 1994). Interestingly, indirect evidence suggests... 

Sullivan, C. H., Mather, I. H., Greenwalt, D. E. and Madara, P. J. 1982. Purification of xanthine oxidase from the fat-globule membrane of bovine milk by electrofocusing. Determination of isoelectric points and preparation of specific antibodies to the enzyme. MoL Cell. Biochem. 44, 13-22. 

The Bik glycoprotein has an isoelectric point (p/) of 2.1 and is composed of a peptide and two glycoconjugate portions [30]. The predicted peptide sequence of Bik is helpful to understanding protein function. The peptide has Kunitz-binding domains I and II attached to the N-terminal peptide tail [31-33] (Fig. 2). Protein mass spectrometry by surface-enhanced laser desorption ionization (SELDI) in combination with detection by Bik antibodies has demonstrated that substantial variation exists in the Bik molecule [13,14]. 

Isoelectric precipitation and acid precipitation are also used to separate antibodies. Isoelectric precipitation (also called euglobulin precipitation) uses the solubility properties of a protein near its isoelectric point.69 When a concentrated protein solution in a low ionic strength buffer is titrated to its isoelectric point, it precipitates very slowly. Table 4 shows a balance of a process for purification of murine monoclonal IgM antibodies.70 The success of precipitation can be followed by the cumulative IgM content in the centrifugate and the sediment. The procedure is very gentle, but very sensitive to environmental conditions. 

For the adsorption of antibodies, virtually all ion-exchangers compatible with proteins can be used. Anion-exchangers have been used extensively for the isolation of polyclonal IgG from human plasma and from that of different mammals in a single step with a high degree of purity in a single pass, as shown in Fig. 8. They have also been used to isolate isoforms of monoclonal IgG after optimization of adsorption and elution conditions to fit with isoelectric point properties of the antibody.91... 

Phosphate buffers are generally used for the chromatography their initial concentration is close to 1 mM and the pH, between 6.5 and 6.9, remains constant. Elution of antibodies is obtained by phosphates, citrates, and fluorides. The most common way to desorb is, in fact, to apply a gradient of phosphate buffer at constant pH. When an antibody with a high isoelectric point is applied, the resolution power of phosphate may not be sufficient enough to separate albumin from the antibody.123... 

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