Serum proteins, separation

Figure 2 illustrates serum protein separation by centrifugal precipitation chromatography the chromatographic tracing of the elution curve in Fig. 2a and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of separated fractions in Fig. 2b. In this example, lOOmL of normal human serum (pooled) was diluted to 1 mL and introduced into the separation channel. The experiment was initiated by filling both upper and lower channel with 75% AS solution followed by sample...

This cross-axis CPC provides the universal application of protein samples with a dextran-PEG polymer-phase system. Using a prototype of the L cross-axis CPC with a 130-mL column capacity, profilin-actin complex was purified directly from a crude extract otAcanthamoeba with the same solvent system as used for the serum protein separation earlier. The sample solution was prepared by adding proper amounts of PEG 8000 and dex-tran T500 to 2.5 g of the Acanthamoeba crude extract to adjust the two-phase composition similar to that of the solvent system used for the separation. The experiment was performed by eluting the upper phase at 0.5 mL/min under a high revolution rate of 1000 rpm. The profihn-actin complex was eluted between 60 mL and 84 mL fractions and well separated from other compounds. The retention of the stationary phase was 69.0% of the total column capacity. 

Roudiere L, Boularan AM, Bonardet A, VaUat C, Ciistol JP, Dupuy AM. Evaluation of a capillary zone electrophoresis system versus a conventional agarose gel system for routine serum protein separation and monoclonal component typing. Clin Lab 2006 52 19-27. 

In CZE, serum proteins have been separated using different buffers (e.g., Tris and Tricine), but mostly borate, with pH of 8-11 [22,25-29]. Serum protein separation can be completed by CE in about 2-10 min in contrast to 1-2 h for agarose electrophoresis (AG) (Figure 26.1). The correlation coefficient between CE and AG for the separated bands is good [30-32]. Some commercial instruments use multicapillaries of narrow diameter (25 p.m) to increase the throughput of the analysis. The narrow capillaries produce better resolution than the wider capillaries with a much shorter migration time [28]. This is true for all CE separations. 

Fig. 4 illustrates serum protein separation by centrifugal precipitation chromatography the chromatographic tracing... 

Separation methods, multichromato-graphic la 56 Serine la 246,356 lb 132 Serotonin la 70,76,239,240,262,355, 380 lb 37-39,231,243,348 Serotonin metabolites lb 327 Serum lipids la 89 Serum proteins la 74 Sesquiterpene derivatives lb 239,446 Sesquiterpene esters lb 239 Sesquiterpene glucosides la 327 Sesquiterpene lactones lb 448 Sevin lb 387-389 Si 50 000, specific surface area la 91 Silica gel, caffeine-impregnated la 85 -, surface modified la 3 Silica gel 60, specific surface area la 91... 

If serum protein or surfactant is added to the acceptor wells, then, in general, p[A l> and P r> are not the same, even under iso-pH conditions. The acceptor-to-donor permeability needs to be solved by performing a separate iso-pH assay, where the serum protein or surfactant is added to the donor side, instead of the acceptor side. The value of Pe is determined, using Eq. (7.20), and used in gradient-pH cases in place of P A /) , as described in the preceding section. The gradient-pH calculation procedure is iterative as well. 

Electrophoresis has also been employed to separate neomycin from analytically-interfering substances such as proteins. Hence Brammer and Hemsonl82 have determined the neomycin content of blood serum. Neomycin was separated from the serum proteins by electrophoresis on cellulose acetate and assayed colorimetrically following elution from the support. 

Proteins have, to date, only rarely been purified by SMB. The first attempt was made by Huang et al. in 1986 [42]. They isolated trypsin from porcine pancreas extracts using an SMB made of only six columns. In addition, this example also demonstrates that SMB systems with a very limited number of columns can be efficient. Another example for a successful protein-separation by SMB is the purification of human serum albumin (HSA) using two SMB-systems connected in series [43]. The first SMB was used for removing the less strongly retained components and the second one for removing the more strongly retained components of the sample matrix. 

Figure 3.27 Polyacrylamide gradient gel electrophoresis of human serum proteins. The proteins are separated in a gel which has an increasing concentration gradient with a parallel decrease in pore size, which restricts the movement of the larger molecules. Note the large number of different protein bands that can be demonstrated. (Photograph by permission of Dr D. Brocklehurst, Department of Clinical Chemistry, Doncaster Royal Infirmary, UK.)...

It is important to realize that the use of these different media for the same sample will result in separation patterns that cannot be easily compared with one another. The separation of serum proteins on cellulose acetate will result in 5-7 bands, while the use of polyacrylamide gel will give 17 bands. 

Figure 11.15 Immunoelectrophoresis of human serum proteins. The proteins are separated electrophoretically from wells cut in a suitable gel. After electrophoresis, a trough is cut in the gel parallel to the direction of migration and filled with an antiserum. The components are allowed to diffuse for 24-48 hours for precipitation lines to develop. Human serum contains many proteins, among which the immunoglobulins can be identified.

Figure 3.23 Selectivity of phenyl and alkyl bonded stationary phase materials for protein separation. Column A, TSK gel phenyl-5PW RP, 75 mm x 4.6 mm i.d. B, TSK gel TMS 250, 75 mm x 4.6 mm i.d. eluent, 60 min linear gradient elution from 5% of 0.05% trifluoroacetic acid in 5%> aqueous acetonitrile to 80% of 0.05% trifluoroacetic acid in 80% aqueous acetonitrile flow rate, lml min-1 detection, UV 220 nm. Peaks 1, ribonuclease 2, insulin-, 3, cytochrome c 4, lysozyme-, 5, transferrin-, 6, bovine serum albumin-, 1, myoglobin-, and 8, ovalbumin.

With the study of the migration of hydrogenium ions (H ) in a phenolphthalein gel by Lodge in 1886 and the description of the migration of ions in saline solutions by Kohlraush in 1897, a basis was set for the development of a new separation technique that we know today as electrophoresis. Indeed, several authors applied the concepts introduced by Lodge and Kohlraush in their methods and when Arne Tiselius reported the separation of different serum proteins in 1937, the approach called electrophoresis was recognized as a potential analytical technique. Tiselius received the Nobel Prize in Chemistry for the introduction of the method called moving boundary electrophoresis. ... 

Magnesium speciation (Section ni.A) in serum was carried out using an anion exchange column for protein separation, with mobile phase at pH 7.4 the effluent was collected in an automatic fraction collector. On-line quantitation of the protein fractions was carried out by DA-UVD, and Mg determination was carried out from the automatic sampler in a GFAAS apparatus, measuring at 202.8 nm . ... 

In 1956, Smithies and Poulik first used 2-DE combining paper and starch gel electrophoresis to separate serum proteins. Nearly 20 years later, polyacrylamide was applied as a support medium. Charge-based protein separation followed as isoelectric focusing (IEF), applied to SDS-PAGE. Later, urea and nonionic detergents were used in IEF-2DE. The most significant achievement was the separation of proteins from E. coli. 

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