Detection of laccase activity in polyacrylamide gels after electrophoresis under denaturing conditions

Summary Specific laccase activity was detected on SDS-PAGE using 2,2-Azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid), guaiacol and syringaldazine as substrates. Enzyme activity was detected immediately following electrophoresis, after the detergent diffused from the gel into a renaturation buffer and subsequent Coomassie blue staining. Identification of laccase in a protein mixture and estimation of its molecular weight were done simultaneously.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

An improved method for arylsulfatase A detection on polyacrylamide slab gels.

An improved method for the detection of arylsulfatase A on polyacrylamide slab gels has been developed using a four-step reaction sequence which leads to the formation of a stable, localized, visible product. The reaction sequence involves the formation of nitrocatechol which, in turn, reduces cupric ferricyanide to form Hatchett's brown, an insoluble brown compound. The next steps involve polymerization of 3,3′-diaminobenzidine and osmication of the polymer. Since the initial reaction product, nitrocatechol, reacts quickly with the cupric ferricyanide to form the Hatchett's brown precipitate, little diffusion occurs and the observed bands of Hatchett's brown are highly localized. The formation of the Hatchett's brown can be observed as the arylsulfatase A reaction proceeds. The final osmium-stained product is very stable and has been stored for weeks without appreciable loss of light absorption or increase in band width. Analysis of arylsulfatase A activity in human leukocyte lysates after discontinuous electrophoresis in polyacrylamide slab gels demonstrated the presence of four bands of activity.     

A method for the direct measurement of glycogen synthase activity on gels after polyacrylamide gel electrophoresis

A method for the detection of glycogen synthase activity after nondenaturing polyacrylamide gel electrophoresis is described. After the electrophoretical run, the gels were incubated in situ with UDP-glucose and glycogen. Labeled or unlabeled UDP-glucose could be used, since similar activity patterns were obtained by autoradiography or iodine staining of the gels. The method here described offers several advantages in terms of speed, sensitivity, and economy when compared with other procedures.     

Detection of DNA and RNA polymerase activities in situ following electrophoresis in polyacrylamide gels.

A procedure is described for the detection of DNA dependent DNA and RNA polymerase activities in intact polyacrylamide gels that contain DNA. After electrophoresis under non-denaturing conditions, the intact gels are incubated with DNA or RNA polymerase reaction mixture in which one of the four deoxyribonucleoside or ribonucleoside triphosphates is radioactively labeled. The acid insoluble radioactivity associated with the intact gel is then analyzed by autoradiography of the intact gel or by liquid scintillation spectrometry of the sliced gel. Inhibition of the enzymatic activities by low molecular weight compounds such as N-ethylmaleimide or rifampin can be demonstrated by this procedure.     

Molecular-sieve chromatography and electrophoresis in polyacrylamide gels

1. The absolute electrophoretic mobilities of eight proteins have been measured at pH8.76, I 0.05, in polyacrylamide gels of 20 different compositions at 10 degrees C. 2. The partition coefficients of these proteins have been determined chromatographically under the same conditions by using columns of granulated polyacrylamide gel prepared simultaneously. 3. The electrophoretic mobilities are an exponential function of the gel concentrations when the latter are corrected for water uptake. The constants of this function have been determined by curvefitting methods. They have been shown to be related to the free solution mobility and to the mean molecular radius respectively. 4. The reduced mobilities have been shown to be a linear function of the partition coefficients by statistical analyses. 5. The physical significance of the relation between electrophoretic mobility and chromatographic phase distribution in gel media is discussed in the context of these results.     

Detection of glutathione transferase activity on polyacrylamide gels

A simple and sensitive assay for glutathione transferase activity on polyacrylamide gel is described. The method is based on the fast reduction of nitroblue tetrazolium salt by glutathione. Blue insoluble formazan colors the gel except in the glutathione transferase area. The stable and defined colorless zone is still detectable with 0.005 unit enzyme. This technique has been successfully applied with enzyme preparations of human heart and other tissues.     

Field inversion gel electrophoresis in denaturing polyacrylamide gels.

The velocities of single stranded DNA molecules in denaturing polyacrylamide gels during symmetric and asymmetric field inversion were measured at different pulse times and gel concentrations. Under the conditions chosen in our study, pulse times as short as a few milliseconds lead to a retardation of DNA molecules larger than 400 bases. We found that a field inversion with an electric field in the forward direction of about double the strength of that applied in the backward direction is a good compromise between the degree of retardation, the temperature control requirements and the run time of the gel.     

Detection of copper on polyacrylamide gels.

A color test for the localization of copper on polyacrylamide gels is described. The test is based upon the quenching of fluorescence of bathocuproine sulfonate by Cu¹⁺ and is sensitive to 0.1 nmol of free or protein-bound copper. There is no false positive reaction with 10 nmol of hemeprotein, free Fe³⁺, Fe²⁺, Co²⁺, or Mn²⁺.     

The instantaneous monitoring of polyacrylamide gels during electrophoresis.

The advantages of being able to see protein zones in a gel during electrophoresis (and hence before staining) are pointed out, and a method is described which depends on local increments of refractive index in these zones. The use of local increments of refractive index in polyacrylamide gels for measuring protein concentrations in zones during electrophoresis is briefly considered; it is found that such increments are greater than would be expected from the amount of protein when sodium dodecyl sulphate is present. The enhancement depends on conditions and time of running. This makes quantitative estimates difficult, but the sensitivity of detection of protein zones by observations based on refractive-index changes is greatly increased by this property of sodium dodecyl sulphate. Methods are described for making optically uniform gels (both with uniform and with graded concentrations of polyacrylamide), necessary for observation of small changes in refractive index. A simple dark-field system of observation is described. Examples are given showing protein samples observed with the system during electrophoresis and compared with the same gel stained with Coomassie Blue after completion of the run. Under optimal conditions the optical method is comparable in sensitivity with staining. With the proteins of lower mol.wt. (approx. 15000), the optical method is not so sensitive, becoming less sensitive with longer running time. This loss of sensitivity is greatly decreased by using more concentrated polyacrylamide gels, and graded gels are therefore more suitable for optical observation than are uniform gels. The observation of protein zones during electrophoresis adds nothing to the time needed for making a stained gel and gives much information long before it can be obtained from the stained gel.     

The electrophoresis of transferrins in urea/polyacrylamide gels.

The denaturation of transferrin by urea has been studied by (a) electrophoresis in polyacrylamide gels incorporating a urea gradient, (b) measurements of the loss of iron-binding capacity and (c) u.v. difference spectrometry. In human serum transferrin and hen ovotransferrin the N-terminal and C-terminal domains of the iron-free protein were found to denature at different urea concentrations.     

Specific staining of myo-inositol-1-phosphatase on polyacrylamide gels after electrophoresis

A sensitive staining method was developed to localise the activity of myo-inositol-1-phosphatase on Polyacrylamide gels after electrophoresis. The method can also be used for non-specific phosphatases as well as for those specific phosphatases acting upon inositol polyphosphates which are prime cellular second messengers. One or two nmol of phosphate is sufficient and less than 3 μg of purified protein will facilitate the localisation of phosphatase. If more phosphatases are present in the enzyme preparation, a combination of inhibitors can be used to suppress the activities of unwanted phosphatases and the use of specific substrates will facilitate the localisation of enzyme of interest. For nomenclature of myo-inositol phosphates recent recommendations are followed. ReferBiochem. J.,258, 1–2 (1989) andEur. J. Biochem.,180, 485–486 (1989). L-myo-inositol-1-phosphate is presently otherwise called as D-myo-inositol-3-phosphate. Dedicated to Dr. F. Eisenberg Jr., on his 70th birthday.     

Steroid-receptor quantitation and characterization by electrophoresis in highly cross-linked polyacrylamide gels.

Conditions for discontinuous polyacrylamide gel electrophoresis have been defined in which progesterone receptors of chick oviduct cytosol and a variety of steroid-binding proteins from other sources are stable and amenable to quantitative analysis. The essential modifications from standard procedures include the use of (1) separation gels in which the cross-linking agent/acrylamide monomer = 15:85, (2) glycerol (10% v/v) in all phases of the Trisglycine-HCl buffer system (pH 10.2 in the separation phase during electrophoresis at 0 degrees), and (3) a layer of a charged reducing agent, thioglycolate, beneath the sample layer. Electrophoresis of untreated oviduct cytosol labeled with [3H]progesterone +/- competing steroids revealed a heterodisperse slow peak and a sharp fast peak. Both peaks displayed the steroid-binding specificity and saturability that are characteristic of intracellular receptors. Recovery of steroid from both the slow and fast components increased linearly with sample load up to 60 mul of cytosol (1.2 mg of protein)/gel (6 mm diameter). The specific progesterone binding detected by this technique was comparable to that detected by charcoal-dextran treatment or ion exchange filtration. Relative electrophoretic mobilities (Rf) of globular protein standards and steroid-protein complexes in cytosol and chick serum were measured in separation gels with total gel concentrations (T) systematically varied from 5 to 15% (w/v). Data were processed by computer programs to obtain weighted linear regressions of log Rf on T (Ferguson plots) and the joint 95% confidence limits of the slopes (-KR) and intercepts of these plots. Molecular radii (R) of the binding components and apparent molecular weights (M) were calculated from the linear correlation of R with KR 1/2 for the standards. The value of M is approximately 158,000 obtained for the cytosol fast component was independent of the length of the separation gel, the presence of a stacking gel or prior exposure of the cytosol to KCl. It was higher than expected from the sedimentation coefficient of 4.2 S in the same pH 10.2 buffer. Electrophoresis in 170-mm separation gels without stacking gels revealed that KCl extracts of protamine-precipitated cytosol contain a different receptor form, of lower net negative charge than the cytosol fast form. The results demonstrate the utility of electrophoresis in highly cross-linked gels of several concentrations to discriminate between various receptor forms and steroid-binding components of serum. This method may lead to overestimates of M for highly asymmetric receptor forms.     

Negative detection of biomolecules separated in polyacrylamide electrophoresis gels

Here we describe the protocols for negative or reverse detection of proteins, nucleic acids and lipopolysaccharides separated in polyacrylamide electrophoresis gels. These protocols are based on the selective synthesis and precipitation of a white imidazole-zinc complex in the gel, which is absent from those zones where biomolecules are located. These methods are highly sensitive (1-10 ng of biomolecules per band), very cheap as they use inexpensive, common laboratory reagents (imidazole and a Zn II salt), rapid (less than 20 min after gel washing), robust and simple (two steps). Reverse-stained biomolecules are reversibly fixed in the gel. After brief incubation in a zinc chelating agent, biomolecules can be recovered from the gel with the same efficiency as from unstained gels. In consequence, they are procedures of choice for micropreparative applications. References covering typical applications are included.