SDS-PAGE analysis

For the purification of RHG, which has been described before (6), A. aculeatus was grown in sugar-beet pulp medium for 48 h. From a 2 1 culture containing 3.2 g protein, about 5 mg RHG was obtained after purification. Analysis by SDS-PAGE revealed only one band of about 55 kDa and thus the enzyme appeared to be pure. After N-glycanase treatment and SDS-PAGE analysis, a smaller protein band of about 46 kDa was visible (Fig.l). 

Compounds not eliminated by the counterscreen are re-tested in a secondary assay involving in vitro translation of FF/HCV/Ren mRNA in Krebs-2 extracts using 35S-methionine followed by SDS-PAGE analysis to monitor protein synthesis... 

EFFECT OF SAMPLE PREPARATION ON CE-SDS AND SDS-PAGE ANALYSIS OF RMABS... 

SDS-PAGE analysis of purified FDH revealed two subunits and, in combination with native PAGE, suggested a a2 i structure. Selenium was not released from the protein during denaturing gel electrophoresis and was... 

Fig. 2.2.4.1 SDS-PAGE analysis of recombinant LK-ADH purification. Lane 1 molecular weight standards (kDa) Lane 2 crude extract of recombinant . coli BL21(DE3)/pADH Lane 3 ADH after anion-exchange chromatography.

In order to investigate whether tomatinases from F. oxysporum and F. solani share similar molecular characteristics, F. solani tomatinase was partially purified. Comparative SDS-PAGE analysis of the protein fractions with and without tomatinase activity showed the presence of a 32.5 kDa band in all positive fractions, while this band was absent in fractions without tomatinase activity The apparent molecular mass of tomatinase of F. solani differs from that of F. oxysporum (50 kDa), S. lycopersici (110 kDa) [33], and Botrytis cinerea (70 kDa)[36]. The F. solani tomatinase presents a very low activity compared with F. oxysporum enzyme [35, 89]. Western blot analysis showed that the two enzymes also differ in their immunological characteristics since the polyclonal antibody against tomatinase of F. oxysporum f. sp. lycopersici did not recognize the tomatinase from F. solani. These results suggest that the enzyme from F. solani is a novel tomatinase species. 

Figure 8. Effect of UV light on binding of [3H]-CPZ to canine striatal homogenates. Fluorogram of irradiated (left) and non-irradiated samples (right) after SDS-PAGE analysis. Taken from the Ph. D. Thesis of K. Thermos.

Usually, 5-10 column volumes are sufficient. Monitor pressure at this step. If the lysate is very viscous, the pressure may exceed the recommended value. Reduce the flow rate accordingly. Start with a flow rate of 0.5—1 mL/min if the His-tagged protein does not bind, the flow rate should be reduced. The flow rate may, however, be increased for protein elution. Collect fractions for SDS-PAGE analysis. 

Usually, 5-10 column volumes are sufficient. Collect fractions for SDS-PAGE analysis. 

For expression of BCCP-p53 fusion proteins, we typically found the protein concentration in crude lysates to be 5 mg/mL, and we estimated that BCCP-p53 was present at approx 1% of total soluble protein. When expressing a number of clones in parallel for array fabrication, the Bradford assay can conveniently be done on all clones in parallel using a microtiter plate format. However, it would be laborious to carry out SDS-PAGE analysis on all clones, so typically we assess only a selection of clones in this way, since the absolute expression level is not critical for array fabrication. 

Figure 2 SDS-PAGE analysis of purified HRV14 2A and 3C proteases. Protein samples (—1-2 (ig) were separated by electrophoresis on 16% gels and then stained with Coomas-sie blue. Lanes 1-4 represent the purification ofHRV14 2A protein. Lanes 1, transformed cell lysate 2, urea-solubilized inclusion bodies 3 and 4, 2A protease preparations after Mono Q and Superdex-75 columns, respectively. Lane 5 represents the purified HRV14 3C protease.

Cholesteryl ester transfer protein (CETP) promotes exchange and transfer of neutral lipids such as cholesteryl ester (CE) and TG between plasma lipoproteins [63-65], The function of CETP is illustrated in Fig. 3. CETP is a very hydrophobic and heat-stable glycoprotein with an apparent molecular weight of 74 kDa as determined by SDS-PAGE analysis [66,67], The cDNA from human liver was cloned and sequenced [68], It encodes for a 476-amino acid protein (53 kDa), suggesting that the apparent higher molecular weight is due to the addition of carbohydrate residues by posttranslational modification. 

Fig. 4. SDS-PAGE analysis of BglA treated with N-glycosidase F. Lane S, protein molecular mass standards lane 1, purified secreted BglA (2.4 pg) lane 2, purified secreted BglA (2.4 pg) treated with N-glycosidase F.

Figure 38. SDS-PAGE analysis of the acid-activated tyrosinase (acid-TY) showing the time course of acid activation at pH 3.0.

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