Electrophoresis activity stains

Middle panel Cell wall proteins were isolated, 10 pgm of each resolved by non-denaturing polyacrylamide gel electrophoresis and PGl and PG2 isoforms detected by activity staining. 

Proteinases and proteinase inhibitors in a sample should be analyzed for their sensitivity to SDS concentration before activity staining is carried out. Theiractivity should be measured in the presence and absence of 0.1 % SDS before proceeding with electrophoresis. The author has characterized many proteinases under such conditions without a loss of activity. Most SDS-sensitive enzymes regain their activity if the gel is washed in the buffer used to dissolve the... 

Using quantitative gel electrophoresis (37) and a simple activity stain (37), Blattler examined urease preparations showing evidences of aging changes (38, 38a). When urease, initially electrophoretically homogeneous, was allowed to age in buffer or 50% diol, enzymically active species appeared that had electrophoretic mobilities between the usual bands. These intermediate bands corresponded to a variety of species that differed by approximately 60,000 molecular weight between 240,000 and 480,000, and between 480,000 and 960,000. 

Fig. 10. Hybridization of GAPDH s (a) rabbit (R) and lobster (L) muscle, pig (P) and lobster (L) muscle (b) rabbit (R) and yeast (Y), pig (P) and yeast (Y). The hybrid bands were separated by electrophoresis on cellulose acetate in 50 mM phosphate buffer, pH 7.0, and revealed in (a) by protein staining and in (b) by activity staining (cf 103).

Activity stains are of great importance during the isolation, purification, and characterization of enzymes, since a particular catalytic reaction is involved and the detection of this activity leads to the unequivocal identification of the zone of interest on the electrophoresis gel. Following separation, the gel is removed from the electrophoresis apparatus and is immersed in a minimal volume of a substrate solution. Detection relies on the formation of a colored product by enzyme in the zones containing the enzyme. Examples of activity stains are given in Table 9.2. 

Figure 5. Tyrosinase isoenzyme forms identified in three different breaks (1—3) after native electrophoresis and staining for dopa oxidase activity. A—D represent small pins, large pins, immature, and mature mushroom samples, respectively. (Reproduced with permission from ref. 35. Copyright 1989/. Food ScL)...

The CK isozymes may be separated by electrophoresis and detected by stains for their enzyme activity. CK-MB immunoassays have also been developed for use in humans that occasionally cross-react with some animal species, although these must first be shown to have appropriate cross-reactivity for the species tested. The LD isozymes have been commonly and readily separated by electrophoresis and stained by their enzymatic activity. For both CK and LD isozymes, their absolute activity is inferred from their relative staining on electrophoretograms and determination of total plasma enzyme activity, typically using an automated chemistry analyzer. 

Activity staining for SOD is carried out in the following way After electrophoresis the gels are immediately soacked in 2,45 x 10 M nitroblue tetrazolium for 20 minutes, followed by an immersion for 15 minutes in a solution of 28 mM TEMED (tetramethylethylendiamine) and 2,8 x 10 riboflavin in 36 mM potassium phosphate pH 7.8. Finally the gels are exposed to an UV-light source and illuminated for 15 minutes. Superoxide dismutase bands appear as colourless zones on the blue gels. 

PPase activity was measured as described in (11). Native polyacrylamide gel electrophoresis was carried out by the procedure described in (12). The activity staining of the gel for PPase was performed as follows. To localize the PPase activity the gel was incubated for 30 min at room temperature in a solution identical with that utilized... 

Intact chloroplasts, pellet and supernatant from osmotically shocked Intact chloroplasts were loaded on a native gel and the electrophoresis was run under low current In a cold room. After separation the gel was treated for activity staining. As shown In Fig. 1 the PPase activity from supernatant (stroma) formed a single band, whereas the PPase activity from pellet and from Intact chloroplast formed two bands. The well washed thylakolds mainly show the lower band. 

For affinity electrophoresis varying concentrations of a polysaccharide fraction which had been isolated from cotyledons were immobilized in a polyacrylamide gel. Equal volumes of a pea leaflet extract were applied to each tube. Following electrophoresis and phosphorylase activity staining, the migration distance of each phosphorylase form, relative to that of bromophenol blue, was determined (Fig. 1). At increasing polysaccharide concentrations the migration velocity of the cytosolic phosphorylase isozyme decreased markedly whereas the retardation of the two chloroplast isozymes was minor. This indicates that the cytosolic phosphorylase isozyme exhibits a much higher affinity towards the immobilized polysaccharide than the plastidic enzyme forms. 

Mailloux, R. J. Singh, R. Appanna, V. D. In-gel activity staining of oxidized nicotinamide adenine dinucleotide kinase by blue native polyacrylamide gel electrophoresis. Anal. Biochem. 2006, 359, 210-215. 

Lin, C. L. Chen, H. J. Hou, W. C. Activity staining of glutathione peroxidase after electrophoresis on native and sodium dodecyl sulfate polyacrylamide gels. Electrophoresis 2002, 23, 513-516. 

Fig. 4. — Monitoring of the Multiple Molecular Forms of Tomato Pectinesterase by Starch-gel Electrophoresis.98 [ENZ, detection of pectinesterase activity by paper print with pectin and Bromothymol Blue PROT, protein staining with nigrosin O, origin. Key A, 1 crude tomato extract after ammonium sulfate salting-out, and dialysis 2 pectinesterase fraction from column of DEAE-Sephadex A-50 3 and 4 pectinesterase fractions from column of Sephadex G-75. B, Two parts of the same gel after horizontal slicing 1, 500 fig of the isolated form of pectinesterase from a column of CM-Seph-adex C-50 with 175 mM phosphate-sodium chloride buffer 2, active fraction at 150 mM buffer 4 and 5, 250 fig and 1 mg of the isolated form of pectinesterase, respectively.]...

FIGURE 11.2 Inhibitory effect of halichlorine on NF- activation in BAECs. (A) BAECs were preincubated with or without halichlorine ( ) for 2h and then exposed to LPS (3 ig/ml) for 3h. Next, cells were collected and then analyzed by RT-PCR. Agarose gel electrophoresis demonstrates mRNA encoding GAPDH, VCAM-1, ICAM-1, and E-selectin. (B) BAECs were preincubated with or without halichlorine ( ) for 2h and then exposed to LPS (3pg/ml) for lh. The p65 subunit of NF- was immunostained with Alexa Flour 568-labeled antibody (red) and nuclei was stained with DAPI (blue). This figure is sited from Tsubosaka et al., 2010b, and is permitted for use by the publisher. 

Fia. 14. Polyacrylamide gel electrophoresis at various purification steps. Gels 1 to 4 were stained for acid phosphatase activity gel 5 was stained for protein. A current of 4 mA/gel was applied to gels 1-3 for 2 hr and to gels 4 and 5 for 6 hr. Gel 1, homogenate 2, DEAE-cellulose peak II 3, DEAE-cellulose peak I 4 and 5, crystalline enzyme. From Igarashi and Hollander (4). 

Fig. 7. Activity and folding of the cell-free produced polypeptides. Autophosphorylation activity of fixsArabidopsis protein kinases. Sodium dodecyl sulfide-polyaciy-lamide gel electrophoresis and Coomassie Brilliant Blue-stained gel of the partially purified products marked with asterisks (A), and the autoradiogram (B). Lanes 1-6 represent Atlg07150, At5g49760, At2g02800, At5g62710, and At4g35500, respectively. NC denotes samples from the reaction mixture incubated in the absence of mRNA. M protein size marker. Note that product of Atlg07150 (lane 1) did not show activity.

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