Activity staining

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. 

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). 

Fig. 3 SDS-PAGE Photograph Separation (Lane Mr and A) and activity staining (Lane B and C) of the crude filtrate of Funalia trogii. Lane Mr standard molecular weight markers ([3-galactosi-dase, 118.0 kDa bovine serum albumin, 79.0 kDa ovalbumin, 47.0 kDa carbonic anhydrase, 33.0 kDa P-lactoglobulin, 25.0 kDa and lysozyme, 19.5 kDa). Relative mobilities of the standard markers vs. common logarithms of their molecular masses were plotted.With the linear regression output, the molecular masses of the proteins in the crude filtrate were estimated (taken from [18])...

The reaction was monitored by TLC (eluent //-hexane diethyl ether, 8 2). 2-Isobutylidene-l-tetralone (UV active) stained light purple with p-anisalde-hyde dip, R 0.66 and the epoxide (UV active) dark purple, R 0.30. 

The reaction was monitored by TLC (eluent petroleum ether ethyl acetate 75 25). The methyl acetoacetate was UV active, stained yellow with p-anisaldehyde, R 0.5. No starting material remained after 48 hours. 

Figure 3.15 Hydrogenase synthesis in regulatory mutants of R. eutropha. Cells were grown on agar plates containing glycerol as the carbon source either in the presence or in the absence H2.The cell material was transferred to filter paper and a triphenyl tetra-zolium chloride-based hydrogenase activity staining was performed. Dark colour reflects activity of the MBH.

A further method to visualize the presence of internalized bacteria is through the use of glucuronidase (GUS) activity stain. The GUS stain is based on the cleavage of a chromagenic substrate (e.g., 5-bromo-4-chloro-... 

Figure B3.1.2 Native discontinuous polyacrylamide gels activity stained for proteinases. (A) Gel stained with Coomassie brilliant blue for total protein. (B) Gel assayed for proteinase activity using casein as a substrate. Samples are enzyme extracts of hepatopancreas from four shrimp species. Lane 1, molecular weight markers Lane 2, Rcaliforniensis Lane 3 R vannamei Lane 4, Rpaulensis, Lane 5, P. schmitti.

Proteinase-containing samples are incubated with a variety of class- or enzyme-specific proteinase inhibitors, separated on a polyacrylamide gel, and activity stained as described in Basic Protocol 3. A clear zone will be evident in lanes where the proteinase is active (i.e., in the absence of inhibitor or in the presence of a mismatched inhibitor). This clear zone will be absent in the lane containing the properly matched proteinase inhibitor, which provides information about the class or type of proteinase detected in the band. 

Techniques have also been developed for the specific visualization of particular classes of enzymes following electrophoretic separation in a gel. These techniques are often referred to as activity staining, as the intrinsic activity of the enzyme is used, either to produce a colored product or to produce a clear zone on a colored background within the gel. A method for visualizing proteinases based on the work of Gar-cfa-Carreno and Haard (1993) and Garcia-Car-reno et al. (1993) is presented (see Basic Protocol 3). 

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. 2. Treatment of cell cultures with deoxymannojirimycin (DMM) inhibited processing of the complex carbohydrate moiety of the epsi-APase but did not affect excretion into the medium. Three-day-old cells grown —Pi as previously described (Goldstein etal., 1988a) were treated with 0.1 mM DMM for 24 h which resulted in inhibition of processing of the complex carbohydrate moiety of the epsi-APase. Culture-medium proteins from +DMM or -DMM treatments were precipitated with 50% acetone, separated via SDS-PAGE and activity stained (also as previously described, Goldstein etal., 1988b). As shown here, inhibition of carbohydrate processing caused a visible increase in apparent molecular mass but did not inhibit the excretion of epsi-APase into the medium.

The Appendix at the end of this chapter lists buffer formulations for stains and 20 enzyme activity staining protocols that have been adapted... 

Appendix Stain Buffer Formulations and Enzyme Activity Stain Protocols... 

The enzyme activity stains presented here are organized according to enzyme function (oxidoreductase, transferase, etc.). Many of the chemicals used in the staining process are toxic and must be handled and disposed of with caution. 

Fig. 8.1. Screening for penicillin G acylase activity. A) Screening in agar plate formats using 6-nitro-3-(phenylacetamido)-benzoic acid (NIPAB) [106], Colonies secreting Penicillin G acylase activity stain a NIPAB-filter yellow. B) Screening in solution using phenylacetyl-MCA and periplasmic extracts without (open symbols) or with ) penicillin G acylases from Kluyvera citrophila, Proteus rettgeri and Escherichia coli respectively [65],...

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. 

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