Electrophoresis, SDS-PAGE

PGE was isolated as desribed in Material and Methods. SDS-PAGE electrophoresis of purified protein showed a single band migrating at approximately 60 kDa. This observation is not in the agreement with the calculated molecular weight of 35 584. However a similar effect has been observed previously in case of PGI and PGC. Apart of the N-glycosylation which plays role in all PGs (Fig. 3), O-glycosylation may also be present as indicated by the band size shift after a treatment of PGE with O.IM NaOH (data not shown). 

EMSA assays can also be exploited to measure STAT nuclear localization, which is, similar to NFkB localization, a measure of STAT activity. Determination of JAK phosphorylation is carried out by immunoprecipitation of the JAK proteins from cell lysates, followed by SDS-PAGE electrophoresis, immunoblotting with antiphosphotyrosine antibody and JAK-specific antibody re-probing [99]. 

Compounds 40—43 interacted with bovine brain-calmodulin as detected in a SDS-PAGE electrophoresis. Calmodulin treated with the lactones had lower electrophoretic mobility than untreated calmodulin. The effect was comparable to that of chlorpromazine, a well known calmodulin inhibitor. In addition, different concentrations of compounds 40 and 41 inhibited calmodulin-depen dent PDEl. The inhibitory activity of herbaru-mins 1 (IC50 = 14.2 xM) and II (IC50 = 6.6 xM) was higher than that of chlorpromazine (IC50 = 9.8... 

SDS-PAGE electrophoresis buffer—Dissolve 30 g of Tfis free base, 188 g of glycine, and 10 g of SDS (gently to avoid foaming) in 9 hters of distilled water. Adjust the pH to 8.3 with concentrated HC1, and dilute to 10 hters with distilled water. 

Reichenbecher, W., R.diger, A., Kroneck, P. M. H., and Schink, B., 1996, One molecule of molybdopterin guanine dinucleotide is associated with each subunit of the heterodimeric MooFeoS protein transhydroxylase of Pelobacter acidigallici as determined by SDS/PAGE electrophoresis and mass spectrometry, Eur. J. Biochem. 237 4139419. 

Fig. 2. (A) SDS-PAGE electrophoresis showing the polypeptide subunits of the reaction-center of Rb. sphaeroides R-26 (B) and (C) SDS-PAGE electrophoresis patterns of the LM-complex and the H-subunit obtained by treating the reaction center with SDS/ LDAO followed by centrifugation in a sucrose-density gradient. Figure from Okamura, Steiner and Feher (1974) Characterization of reaction centers from photosynthetic bacteria. I. Subunit structure of the protein mediating the primary photochemistry in Rhodopseudomonas sphaeroides R-26. Biochemistry, 13 137, 138.

At present the most sensitive indicators of effect arey aM-U and RBP-U, both belonging to the low molecular mass proteins (Herber et al.. 1988). Enhancement of started at a Cd-U concentration of 3-6 molecular mass proteins are used for the assessment of tubular damage, the determination of RBP-U is to be preferred over /SaM-U, as the latter protein already deteriorates in the bladder at pH 5.5. For the determination of / aM-U. RBP-U and also Alb-U a standardized latex immunoassay method is available (Herber et al., in press). Another possibility to assess tubular proteinuria and glomeruiar proteinuria is SDS-Page electrophoresis (Herber et al., 1988). 

SDS-PAGE electrophoresis was used to analyse P40 during its purification and monitor its refolding. P40 exhibited different electrophoretic mobilities when it was boiled or not in the presence of SDS (Fig. 10). 

Figure 10. Analysis of purified P40 by SDS-PAGE electrophoresis. Proteins (lOpg) were separated on a 12.5% acrylamide gel before staining with Coomassie blue. Lanes 1 and 6, molecular mass markers (kDa) lane 2, P40 non reduced and non heated lane 3, P40 reduced and non heated lane 4, P40 non reduced and heated at 100°C for 10 min in sample buffer lane 5, P40 reduced and heated at 100°C for 10 min in sample buffer.

SO that in the second batch productivity was severely reduced and conversion yield was significantly lower even at prolonged operation. SDS-PAGE electrophoresis revealed that most of the desorbed protein had a molecular weight of 31,000 Da, which corresponds to that of Alcaligenes faecalis lipase. Most of the activity lost from batch to batch corresponded to protein desorption from the matrix, enzyme inactivation being quite low. Therefore, it made very little sense to use QLG instead of QL, but the free enzyme was not recoverable from the reaction medium and cost estimates indicated that the enzyme should be used at least five times to make the process economically attractive. Therefore, the next goal was to construct an immobilized lipase biocatalyst from soluble QL. The hydrophobic nature of the active site and the requirement of a hydrophobic interface for lipase action made reasonable to use hydrophobic supports however to test the validity of this hypothesis several immobilization systems were tested. The results obtained are summarized in... 

After subjected to purifying, the CGTase secreted by B. alkalophilus 1177 and the standard proteins were subjected to SDS-PAGE electrophoresis simultaneously. 

According to the SDS-PAGE electrophoresis performed on standard proteins with known molecular weight (Fig. 2.5), standard protein relative mobility was plotted against logarithm molecular weight of standard protein as shown in Fig. 2.6. 

Fig. 2.5. The plot of the SDS-PAGE electrophoresis of pure CGTase from B. alkalophilus and standard proteins [7].

As shown in Fig. 2.13, the SDS-PAGE electrophoresis of CGTase from B. alkalophilus p. G1 showed a single protein band, and by comparing with the standard protein bands, the molecular weight of CGTase was judged to be 75 kDa. 

Fig. 2.13. The SDS-PAGE electrophoresis of pure CGTase standard protein [ 10].

Gambuti, A. Rinaldi, A. Pessina, R. Moio, L. Evaluation of aglianico grape skin and seed polyphenol astringency by SDS-PAGE electrophoresis of salivary proteins after the binding reaction. Food Chem. 2006, 97, 614—620. 

Hgure 9 Canine urine. One-dimensional SDS-PAGE electrophoresis of the proteins in dog urine from two different animals before and after castration. After castration the pattern is similar to that of females. Differences in levels of the male-specific protein (indicated by an arrow) may be associated with different prostate disorders. (Courtesy of Miller I, University of Veterinary Medicine, Vienna.)... 

Lately, the discontinuous Laemmli system buffer (containing 0.1% w/v SDS) is most commonly used for SDS-PAGE. The method has been widely used for the characterization of the protein profiles of processed whey, meat, cereal, legume, and seed proteins (Roy et al., 2007 Miyamoto et al, 2009 Boye et al, 2010b Sikes et al., 2010 Warchalewski and Gralik, 2010) as well as the analysis of membrane proteins (Braun et al, 2009). Further information on SDS-PAGE electrophoresis can be found elsewhere (Amersham, 1999a). 

Figure 14.13 shows the sample protein assay results of using a commericial 100-kDa microdialysis probe (PES dialysis membrane, an effective length 10 mm, 0.4 mm OD) to monitor bovine intervetebral disk (IVD) cultore (Li et al., 2006). The IVD is cultured up to 7 days, and the probe is operated by either push, puU, or push-and-puU modes. Apart from glucose and lactate, the probe picked up at least three proteins with a molecular weight around 40 kDa. Three peaks of proteins (PI, P2, and P3) were evident on fast protein liquid chromatography (FPLC) outcome during the 7-day culture period. Three bands were also evident using 12% SDS PAGE electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis) with silver stain (see Fig. 14.13). In these experiments, the relative recovery is easily determined using the developed in situ calibration using 40-kDa fluoresecent dextran as the internal standard. In the 7-day experiments, the relative loss of fluorescent dextran (40 kDa) stablized at 8 -10%, which is equivalent to the protein transmission across the membrane (Li et al., 2006).

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