Protein mixture

Plant proteins from single sources, such as soybean meal, may be abundant in specific amino acids that are deficient in some cereal grains. Thus a combination of soybean meal and com with theh amino acid symbiosis may provide an exceUent amino acid profile for dogs. Plant protein mixtures alone do not meet the amino acid needs for cats, because taurine [107-35-7] is not generaUy present in plant proteins. 

Generally, optimizing the selectivity by choosing a gel medium of suitable pore size and pore size distribution is the single most important parameter. Examples of the effect of pore size on the separation of a protein mixture are given in Fig. 2.15. The gain in selectivity may then be traded for speed and/ or sample load. However, if the selectivity is limited, other parameters such as eluent velocity, column length, and sample load need to be optimized to yield the separation required. 

FIGURE 2.15 Influence of the pore size of Sephacryl HR on the separation of proteins of various molecular mass. The protein mixture is composed of ferritin, aldolase, ovalbumin, and chymotrypsinogen A. [Reproduced from Hagel et al. (1989), with permission.]... 

The recommended flow rates of 0.5 to 1.0 ml/min for stainless steel and 0.4 to 0.8 ml/min for glass analytical columns will provide adequate resolution of most protein mixtures. 

The hydrophilic surface characteristics and the chemical nature of the polymer backbone in Toyopearl HW resins are the same as for packings in TSK-GEL PW HPLC columns. Consequently, Toyopearl HW packings are ideal scaleup resins for analytical separation methods developed with TSK-GEL HPLC columns. Eigure 4.44 shows a protein mixture first analyzed on TSK-GEL G3000 SWxl and TSK-GEL G3000 PWxl columns, then purified with the same mobile-phase conditions in a preparative Toyopearl HW-55 column. The elution profile and resolution remained similar from the analytical separation on the TSK-GEL G3000 PWxl column to the process-scale Toyopearl column. Scaleup from TSK-GEL PW columns can be direct and more predictable with Toyopearl HW resins. 

FIGURE 7.2 (A) Separation of a standard protein mixture. A test mixture consisting of BSA... 

FIGURE 7.4 Separation of a standard protein mixture on a Fractogel EMD BioSEC-column (600-16 mm) after incubation with 30% acetonitrile. The sample contained BSA ( ), ovalbumin ( ), and cytochrome c (A) (sample volume 500 ftl flow rate 1.0 ml/min). No significant shifts of the retention times and no loss of the resolution were observed even after 900 hr of exposure. 

The chromatogram of the protein mixture should show the partial separation of serum albumin and ovalbumin with a trough of at least 30% of height between their peak signals and baseline separation between ovalbumin and cytochrome c. If present in the sample, the dimeric form of serum albumin should also appear as an individual peak signal before elution of the monomeric form. 

For proteins, the most useful columns are those with pores of 100-500 A, as seen in Fig. 10.2, because most proteins elute on the linear portions of the calibration curves. Figure 10.5 illustrates an analysis of a protein mixture on SynChropak GPC100. Small peptides can be analyzed on the 50-A SynChro-pak GPC Peptide column with appropriate mobile-phase modifications. Many peptides have poor solubility in mobile phases standardly used for protein analysis, as discussed later in this chapter. 

H. Yamamoto, T. Manabe and T. Okuyama, Apparatus for coupled high-performance liquid chromatography and capillary electrophoresis in the analysis of complex protein mixtures , 7. Chromatogr. 515 659-666 (1990). 

Figure 26.2 Separation of a protein mixture by electrophoresis. At pH 6.00, a neutral protein does not migrate, a basic protein is protonated and migrates toward the negative electrode, and an acidic protein is deprotonated and migrates toward the positive electrode.

Biological targets may consist of single-entity proteins, complexes of receptors (dimers), or receptors plus accessory proteins. Mixtures of gene products can produce unique phenotypic biological targets. 

The resolution of these columns for protein mixtures, however, was comparably poor. The peak capacity for human serum albumin was near 3 during 20 min gradient elution. Improvement has been reached by covalent binding of PEI (M = 400-600) onto a 330 A silica of 5 pm particle size [38], The peak capacities of ovalbumin and 2a -arid glycoprotein were 30-40 (tgradienl = 20 min). Enhanced peak capacity and resolution probably were due to the more diffuse structure of PEI coupled to silane moieties than that of strictly adsorbed on silica and cross-linked (see Sect, 2.2). Other applications of covalently adsorbed PEI are discussed in Sect. 4.1. 

Protein mixtures were well resolved on poly(aspartic acid)-silica columns using 0.05 mol/1 phosphate buffer, pH 6.0 and a gradient of sodium chloride from 0 to 0.6 mol/1. The columns displayed a high capacity and selectivity. Figure 3 shows the separation of several standard proteins with isoelectric points ranging from 4.7 to over 11. Peaks are sharp and show minimal tailing. The poly(aspartic acid) coating was quite stable the columns lasted for hundreds of hours of use without decrease in efficiency and capacity. 

Liquid samples might appear to be easier to prepare for LC analysis than solids, particularly if the compounds of interest are present in high concentration. In some cases this may be true and the first example given below requires virtually no sample preparation whatever. The second example, however, requires more involved treatment and when analyzing protein mixtures, the procedure can become very complex indeed involving extraction, centrifugation and fractional precipitation on reversed phases. In general, however, liquid samples become more difficult to prepare when the substances are present at very low concentrations. 

A more typical application of micro-reticulate resins in exclusion chromatography is shown by the separation of a standard protein mixture depicted in figure 3. 

The Separation of a Standard Protein Mixture by Exclusion Chromatography on a Vinyl Alcohol-Styrene Co-Polymer... 

Douglas, NG Humffray, AA Pratt, HRC Stevens, GW, Electrophoretic Mobilities of Proteins and Protein Mixtures, Chemical Engineering Science 50, 743, 1995. 

Noncollagen proteins Plasma proteins Mixture of various plasma proteins. 

FASPEK KIT for buckwheat Soluble peanut protein mixture... 

Dai, Y., Whittal R.M., and Li, L., Two-layer sample preparation a method for MALDI-MS analysis of complex peptide and protein mixtures, Anal. Chem., 71, 1087, 1999. 

Riickert, M., Wohlfarth, M., and Bringmann, G., Characterization of protein mixtures by ion-exchange chromatography coupled on-line to nuclear magnetic resonance spectroscopy, ]. Chromatogr. A, 840, 131, 1999. 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

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