Protein bands, detection

The conformational change of the carrier in relation to the state of the loops facing the matrix was examined in more detail by studies on the effect on cysteine residues of copper-o-phenathroline(Cu(OP)2), which catalyzes disulfide bond formation between SH-groups [11]. Incubation of the particles with Cu(OP)2 specifically induced formation of a 60 kDa protein band detected by SDS-PAGE with decrease in the 30 kDa carrier band (Fig. 8), but no change in the protein band of the carrier in mitochondria (data not shown). The 60 kDa protein was reversed to the 30 kDa carrier by treatment with 2-mercaptoethanol (data not shown). Therefore, Cu(OP)2 specifically catalyzed the formation of an S-S bond between cysteine residues of the carrier acting from the matrix side. As the ADP transport... 

Figure 52-3. Diagrammatic representation of the major proteins of the membrane of the human red blood cell separated by SDS-PAGE. The bands detected by staining with Coomassie blue are shown in the two left-hand channels, and the glycoproteins detected by staining with periodic acid-Schiff (PAS) reagent are shown in the right-hand channel. (Reproduced, with permission, from Beck WS, Tepper Ri Hemolytic anemias iii membrane disorders, in Hematology, 5th ed. Beck WS [editor]. The MiT Press, 1991.)...

Figure 5 shows the pattern of lyase isoenzymes along the purification process at first, three bands with lyase activity (pis 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1) in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pi 9.20 (B 2), but more proteins were detected by silver staining (A 2). 

However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3). 

Results of electrophoretic analysis of proteins in the allantoic fluid and blood serum before and after photodynamic treatment are presented in Figs. 5.5 and 5.6. Visually, we did not detect any changes in the position and intensity of protein bands. In order to quantitatively analyze these parameters we scanned the gels and measured the relative optical density of the bands (Figs. 5.7 and 5.8). 

After electrophoresis, protein bands are transferred onto a solid support. Many aspects of a transfer can affect antigen detection. Some of these factors are specific to the transfer method and choice of membrane, whereas others apply to the entire blotting procedure. For example, the transfer efficiency may be affected by the presence of SDS in the gel and whether the gel was stained prior to transfer. 

Protein bands were detected with the ECL plus Western Blotting Detection system according the manufacture s protocols. 

Sensitivity of fluorescent stains is very dependent on protein amino acid composition. Minimum amount detected based on amount of protein loaded onto gel. The actual amount on the blot will be slightly lower because of losses during electrotransfer. Values are based on use of a full-sized gel (I I cmx I6cmx 1.5 mm). Sensitivity will be -2 to 5 times higher when minigels (8 cm x 10 cm x 1.0 mm) are used because the protein bands are concentrated on a smaller area of membrane. 

Destain membrane with several changes of water for 30 sec to 1 min each, then air dry. If the stain is to be extracted from protein bands (steps 4 to 6) prior to employing a second detection method, omit drying. 

Do not overdestain because the protein bands will be difficult to detect. Stop destaining when the background has a very slight pink tinge. 

Ideally, the gel should have been stained with a Coomassie stain—preferably a colloidal Coomassie if detection sensitivity is an issue, e.g., (2,3), or one of the commercially available stains. This method is also compatible with Sypro stains (though an ultraviolet transilluminator will be required to visualize the protein bands or spots during excision). Standard silver stains are not compatible, but certain modified silver stains—e.g., (4), or one of the commercially available mass spectrometry compatible silver stains—can be used however, even these usually give inferior mass spectrometry data compared to Coomassie- or Sypro-stained gels. 

The interdependence of protein surface and hydration of Sephadex has implications for confusion of the molecular mass experiments. The acid carboxypeptidase from A. saitoi is a glycoprotein, containing approximately 30% carbohydrate by weight [81, 82], Furthermore, a single protein band with molecular mass of 72,000 was detected in SDS-PAGE [79], This indicates that the carboxypeptidase is a homologous dimmer [79],... 

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