Calibration proteins

TABLE 7.3 Selection of Calibration Proteins Commonly Used for the Calibration of Fractogel EMD BioSEC Columns ... 

The molecular masses of polygalacturonase and exopolygalacturonase were approximately determined by gel chromatography on Superose 12 using FPLC device (Pharmacia, Sweden) and the Calibration proteins II kit (Boehringer-Mannheim, Germany). 

Calculation of the molecular mass of an unknown protein follows the same procedure as, for example, quantitative protein determination plotting of the Rf of the calibration proteins against their molecular mass computing of a standard curve and estimation of the MW of the unknown protein and using the regression functions of the standard curve (c.f Fig. 2.1). 

Ninety-three percent of laboratories submitted their data to either (or both) the ExPASy or Propsearch Internet sites. One laboratory used its own identification software and 4 did not attempt identification. Forty-one facilities reported the analysis of a calibration protein of their choice that was analyzed along with the experimental sample. Both the methods of analysis and search programs contain many variables, and it is necessary that all data be treated similarly to allow comparisons. To allow for comprehensive comparisons, the AAA committee recalculated the mol % and error data as described in Materials and Methods and submitted all data sets to both search programs. For the ExPASy site, data were submitted with and without the calibration protein to determine the benefit of a calibrant. The conclusions drawn below are from the committee s resubmissions. 

Analysis using the ExPASy site was more complex because data could be entered with or without accompanying calibration protein data. When data were entered without calibration proteins, only 30.6% of the entries were identified as tpis rabit, 52.7% were identified as tpis from other species, and 16.67% were... 

Figure 4. ExPASy Scores for tpis rabit When Data Were Submitted without or with Data from a Calibration Protein. Amino acid analysis data were submitted to the ExPASy site using the 16 residue Constellation 2 with (empty bars), and without (filled bars), known protein (calibrant) data furnished by the participants. The chart shows rank assigned to tpis rabit (SwissProt data base) for selected sites (a) Sites (n = 14) where accompanying data improved rank of tpis rabit. (b) Sites (n = 11) where including calibration data degraded the rank obtained for the query protein. For 16 sites (not shown), there was no change in rank of rabbit tpis with the inclusion of calibration data (see Table IV). Rank values above 10 are truncated.

Several of the individual urinary proteins (e.g., albumin, immunoglobulins) can be measured by quantitative immunochemical methods when suitable antisera and calibration proteins are available (Woo et al. 1978 Bernard, Vyskocil, and Lauwerys... 

Fig.3 SDS-PAGE of SOD from Scots pine needles. Molecular weight calibration proteins phosphorylase b (94 kDa) albumin (67 kDa) ovalbumin (43 kDa) carbonic anhydrase 30 (kDa) trypsin inhibitor (20.1 kDa) -lactalbumin (14.4 kDa).

SDS gel electrophoresis pattern of the CFpFj aration and calibration proteins (C al). A small amount of carbosylase can be seen. refer to CF subunits I,II,III,IV to CF subunits. 

PSS-SG composite film was tested for sorption of heme proteins hemoglobin (Hb) and myoglobin (Mb). The peroxidaze activity of adsorbed proteins were studied and evaluated by optical and voltammetric methods. Mb-PSS-SG film on PG electrode was shown to be perspective for detection of dissolved oxygen and hydrogen peroxide by voltammetry with linear calibration in the range 2-30 p.M, and detection limit -1.5 p.M. Obtained composite films can be modified by different types of biological active compounds which is important for the development of sensitive elements of biosensors. 

The preliminary precipitation of proteins from milk is realized through the addition of solutions of acetic acid (1,7 mol/1) and sodium acetate (lmol/1) at t = 40-45°C before chromatographic isolation of OxTC. The precipitated proteins are separated by filtration. OxTC is detenuined in filtrate after its isolation on chromatographic column. Contents of OxTC was determined on calibration curve which is linear within concentration range 0,01-1,0 p.g/ml. 

FIGURE 2.4 Calibration curve of dextran on Sephacryi S-300 SF. Calibration curves were calculated from one chromatogram of a broad MWD reference sample using data for the molecular mass distribution as obtained by a calibrated gel filtration column ( , upper curve) and on-line MALLS ( ). The calibration curve was found useful for estimating the size of globular proteins. [Reproduced from Hagel et al. (1993), with permission.]... 

FIGURE 4.2 Polyethylene oxide, dextran, and protein calibration curves for TSK-GEL SW Columns. Column TSK-GEL SW, two 7.S mm x 60 cm columns in series. Sample , proteins Q, polyethylene oxides O, dextrans. Elution dextrans and polyethylene oxides distilled water proteins 0.3 A1 NaCI in 0.1 M phosphate buffer, ph 7. Flow rate 1.0 ml/min. Detection UV at 220 nm and Rl. 

The ionic species of the mobile phase will also affect the separation. This is shown in Table 4.3 by the difference in resolution values for magnesium chloride buffer compared to sodium sulfate buffer. In addition, calibration curves for proteins in potassium phosphate buffers are shallower than those generated in sodium phosphate buffers. The slope of the curve in Sorenson buffer (containing both Na and ) is midway between the slopes generated with either cation alone (1). Table 4.4 illustrates the impact of different buffer conditions on mass recovery for six sample proteins. In this case, the mass recovery of proteins (1,4) is higher with sodium or potassium phosphate buffers (pH 6.9) than with Tris-HCl buffers (pH 7.8). 

TSK-GEL PW type columns are commonly used for the separation of synthetic water-soluble polymers because they exhibit a much larger separation range, better linearity of calibration curves, and much lower adsorption effects than TSK-GEL SW columns (10). While TSK-GEL SW columns are suitable for separating monodisperse biopolymers, such as proteins, TSK-GEL PW columns are recommended for separating polydisperse compounds, such as polysaccharides and synthetic polymers. 

FIGURE 4.43 Calibration curves for globular proteins on toyopearl resins. Column 22 mm X 30 cm. Sample Protein standards. Elution 0.06 A1 phosphate buffer, pH 7, in 0.06 A1 KCI. Legend elution volume V column volume. 

FIGURE 6.40 Calibration curves of Shodex PROTEiN KW-800 series. Column Shodex PROTEiN KW-802.S, KW-803, KW-804, 8.0 mm i.d. X 300 mm. Eiuent Pullulan, PEG PEO Purified water, Protein SO mM Sodium phosphate buffer + 0.3 M NaCI (pH 7.0). Flow rate 1.0 mL/min. Detector Pullulan. PEG PEO Shodex Rl Protein Shodex UV (220 nm). Column temp. Ambient. 

FIGURE 7.13 Preparative separation of various proteins on Fractogel EMD BioSEC (S). The length of the column was 1000 mm and the inner diameter 100 mm. The flow rate was 6.2 ml/min with 20 sodium phosphate buffer (pH 7.2) containing 0.3 M NaCI as the eluent. The injected standard proteins can be used to create a calibration curve. 

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. 

FIGURE 10.2 Calibration curves for proteins on SynChropak GPC columns. Mobile phase 0.1 M potassium phosphate, pH 7. (From MICRA Scientific, Inc., with permission.)... 

Anionic and neutral polymers are usually analyzed successfully on Syn-Chropak GPC columns because they have minimal interaction with the appropriate mobile-phase selection however, cationic polymers adsorb to these columns, often irreversibly. Mobile-phase selection for hydrophilic polymers is similar to that for proteins but the solubilities are of primary importance. Organic solvents can be added to the mobile phase to increase solubility. In polymer analysis, ionic strength and pH can change the shape of the solute from mostly linear to globular therefore, it is very important to use the same conditions during calibration and analysis of unknowns (8). Many mobile phases have been used, but 0.05-0.2 M sodium sulfate or sodium nitrate is common. 

Gels made in this way have virtually no usable porosity and are called Jordi solid bead packings. They can be used in the production of low surface area reverse phase packings for fast protein analysis and in the manufacture of hydrodynamic volume columns as well as solid supports for solid-phase syntheses reactions. An example of a hydrodynamic volume column separation is shown in Fig. 13.2 and its calibration plot is shown in Fig. 13.3. The major advantage of this type of column is its ability to resolve very high molecular weight polymer samples successfully. 

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