Proteins, calibrant

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. 

Figure 1. Protein calibration curves for the Spherogel TSK-SW 2000 and SW 3000 columns. Ten fiL containing 10--100 fig of each protein in 0,2M KPO buffer (pH 6.8) was chromatographed in the same buffer at 1.0 mL/min. Detection was at 254 nm X 0.16 AUFS pressure 500 psi. A chromatogram of 4 proteins on the SW 2000 column is also shown.

Figure 4.25 Protein calibration curves for Spherogel TSK-SN 2000 and TSK-SM 3000 colunn. Mobile phase phosphate buffer 0.2 M, pH 6.8, flow rate 1.0 sl/sin.

To test this idea, a Bio-Rad Protean II electrophoresis cell and Bio-Rad Model 3000xi computer-controlled power supply were used to carry out the electrophoretic separation and recovery of pre-stained and unstained protein calibration standards (lysozyme, soybean trypsin inhibitor, carbonic anhydrase, ovalbumin, bovine serum albumin and phosphorylase B) obtained from Bio-Rad Laboratories. Standard SDS-gel electrophoresis techniques were used [167]. 

A reference material (RM) for traceability of total protein in human urine does not exist. The control materials used in clinical laboratories for calibration purposes are not unified. Some laboratories use bovine serum albumin as a calibration material (URINE-CHIMIE BIOTROL) [5], whereas others use a mixture of human serum albumin (70%) and globulin (30%) (LYPHOCHECK Quantitative Urine Control, BIO-RAD) [6]. The use of various protein calibration standards yields various results of... 

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.

The calibration of total protein, albumin, and specific protein methods remains a problem when analyzing samples from laboratory animals. Many of the available protein calibration materials are bovine or human in origin, and there are international reference proteins for some human proteins (Whicher 1984 Price and Newman 1997 Tiffany 1999). However, because calibration standards for laboratory animals are not widely available, the values between methods may show wide variations due to differences in calibrators. When some protein fractions are available for laboratory animals, these materials are often less than 95% pure. The investigator therefore... 

Various colorimetric methods are also available, based on non-specific dye binding to polypeptide chains, one of the more common being the Bradford assay. One drawback with such methods is that the actual colour intensity (absorbance) developed is not absolute, but depends on the specific protein. Calibration can therefore be a problem if accurate concentrations are required. 

FIGURE 23.1 Example of a mustard seed protein calibration curve for a sandwich ELISA. (Courtesy of EUsa Systems.)... 

Since we are considering the protein calibration standards in this study to be spheres, we also assume that r is proportional to the cube root of the molecular weight. This leads to the relationship... 

Protein Calibration Standard I (Bruker Daltonics, Wissenbourg, France) insulin, ubiquitin I, cytochrome C, myoglobin. Covered mass range 5,000-17,500 Da. Store at -20°C. 

A protein calibration was performed by using a modified PLS (MPLS) regression of first- and second-derivative spectra. In the validation set a standard error of 0.14% was obtained. This value is considered to be quite high, probably due to the variable nonprotein nitrogen (NPN) contents of samples. The error in protein calibration may occur partly because the reference method includes tme protein and NPN, whereas in NIRS NPN behaves differently from tme protein. 

For example, Figure 8.15 shows the spectra corresponding to the calibrations for protein obtained from the set of wheat data we used for our illustrative examples. These spectra correspond to the use of one, two, and three principal components to develop the calibration equation. The plot of the calibration equation can be examined to determine the characteristics of the spectra that are important to the prediction process. In Figure 8.15, for example, we see positive excursions at 2180 nm (corresponding to the absorbance of the protein amide) and a doublet at 1700 nm (corresponding to the overtone of C—H stretching) therefore these are important contributors to the protein calibration. 

For the preparation of protein calibration curve, bovine serum albumin (BSA) was used. Different concentrations of BSA were prepared as 1 ml, and 2 ml of diluted Bradford reagent were added. The absorbance of these solutions was measured at 595 nm. 

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