Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels

A system was developed for the rapid characterization of microbial pectic enzyme complexes and then tested on Erwinia chrysanthemi and Sclerotium rolfsii. Pectic enzymes in minute samples of crude culture filtrates were resolved by ultrathin-layer polyacrylamide gel isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then assayed with an ultrathin pectate-agarose overlay stained with ruthenium red. The simple procedure can be completed within 30 min after isoelectric focusing, can detect extremely low levels of pectate lyase (6.4 x 10 mumol of product per min), and is sufficiently sensitive to determine the pectate lyase isozyme profile of a single bacterial colony with a diameter of 4 mm. Pectate lyases and polygalacturonases can be distinguished by altering buffer conditions in the overlays. The assay system revealed additional isozymes not resolved by classical techniques and generally corroborated the previously published isoelectric points and molecular weights of the pectate lyase isozymes and exo-poly-alpha-d-galacturonosidase produced by E. chrysanthemi and the endopolygalacturonase and exopolygalacturonase produced by S. rolfsii.     

Evaluation of isoelectric focusing running conditions during two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis: variation of gel patterns with changing conditions and optimized isoelectric focusing conditions

Five major isoelectric focusing (IEF) parameters--volt-hours; concentrations of acrylamide, NaOH, and H3PO4; and equilibration time--were systematically varied to determine the effect of each on two-dimensional IEF/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel patterns and to optimize IEF conditions. Alterations in each parameter affected the gel pattern, frequently causing uncertainty in the identification of spots between conditions. The results emphasize the need for internal analytical consistency, and indicate that gel pattern comparisons between laboratories can be complicated if different IEF conditions are employed. The systematic evaluation indicated that optimized patterns were obtained when increased concentrations of NaOH and H3PO4 (to 50 and 25 mM, respectively) and run durations of 10,000 V-h or longer were used.     

Analysis of immune response: comparison of immunoblots after isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cytoplasmic protein extract from Brucella

Analysis of the immune response towards the facultative intracellular bacterium, Brucella melitensis, was studied by immunoblotting after either isoelectric focusing (IEF) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A cytoplasmic extract (CPE) of Brucella melitensis was used as antigen to analyse the response in 17 sera from naturally infected goats. CPE analysed by IEF exhibited 25 proteins within the pH range of 4.35 to 6. Immunoblotting revealed most of the stained bands around pH 4.5-5.4. CPE analysed by SDS-PAGE showed more than 20 silver stained proteins in the molecular range of 16-18 kDa to 70 kDa but immunoblotting revealed only 1 to 6 bands according to the sera tested. Because proteins are preserved in their native state with IEF, in contrast to SDS-PAGE treatment, this technique may be best suited for analysis of the overall response to natural infection.     

Reverse fibrin autography: a method to detect and partially characterize protease inhibitors after sodium dodecyl sulfate--polyacrylamide gel electrophoresis

A new technique, reverse fibrin autography, was developed to detect protease inhibitors previously fractionated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Exogenous proteases were incorporated into fibrin-agar indicator films, eventually causing the fibrin to lyse. When an acrylamide gel containing inhibitors was placed on top of such an indicator, the positions of the inhibitors were revealed by the formation of opaque, lysis-resistant zones in the otherwise cleared fibrin film. The technique was versatile in that a variety of inhibitors were revealed, and semiquantitative since the size of the lysis-resistant zone in the indicator increased in proportion to the amount of inhibitor subjected to electrophoresis. This approach could be used not only to detect inhibitors having different protease specificities, but also to distinguish between the inhibitor activities of antibodies directed against urokinase or tissue-type plasminogen activator. Thus, reverse fibrin autography offers a convenient new approach to rapidly screen and partially characterize inhibitors present in complex biological samples.     

SDS聚丙烯酰胺凝胶电泳快速染色新方法的研究

通过几种金融盐溶液对SDS聚丙烯酰胺凝胶电泳染色的实验表明,0.25mol/L的CaCl2和MgCl2溶液能够对蛋白质进行有效的染色,经这2种溶液染色的蛋白质都能够从凝胶中洗脱回收。尤其是CaCl2法灵敏度更高,而且蛋白质条带形成之后也十分稳定,所以在运用制备电泳纯化蛋白质时这种新的染色方法较适用。     

Direct detection of an antimicrobial peptide ofPediococcus acidilactici in sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Summary An SDS-PAGE technique is described that allows identification of the antimicrobial activity of a peptide secreted by a strain ofPediococcus acidilactici. This peptide has an antimicrobial property against several baeteria associated with food. This technique enables detection of the specific peptide (or protein) band(s) associated with the inhibitory effect which can then be eluted from the gel for further studies.Published with the approval of the Director, Wyoming Agriculture Experiment Station as Journal Article No. 1526.     

Lysozyme oligomers as a molecular mass standard for sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Egg white lysozyme treated with hypochlorous acid links together producing di-, tri-, tetra-, and pentameric derivatives with molecular masses ranging from 14,300 to 90,500. Similar oligomeric products may be obtained by treating lysozyme color derivatives produced by labeling lysozyme with fluorescein, trinitrobenzenesulfonic acid and 2,4-dinitrofluorobenzene, with hypochlorous acid. The oligomeric lysozyme derivatives thus obtained consist of a mixture of proteins with molecular masses equal to multiples of 14,300 (lysozyme molecular mass). This mixture can be applied as a set of molecular mass standards suitable for determination of protein molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis of protein mixtures containing active or potentially active proteases: Analysis of human exocrine pancreatic proteins

Analysis of human pancreatic juice in two dimensions using isoelectric focusing followed by sodium dodecyl sulfate gel electrophoresis indicated that human pancreatic trypsinogen (IEP_n = 6.4) rapidly autoactivated in the absence of the secretory trypsin inhibitor. The addition of 4 to 6 m urea to the protein sample and 8 m urea to the isoelectric focusing gel inhibited this autoactivation process and allowed the analysis of human exocrine pancreatic proteins. Thirteen discrete proteins were separated by the two-dimensional gel procedure including two forms each for trypsinogen, proelastase, and procarboxypeptidase A, and single forms each for amylase, lipase, procarboxypeptidase B, and chymotrypsinogen. The kinetics of inhibition of human trypsin by 8 m urea in the presence of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid indicated that samples containing active proteases could also be analyzed by this procedure.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing

Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe0-transferrin, Fe1-transferrin and Fe2-transferrin region. At the level of Fe0-transferrin and Fe1-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

New method for analyzing the molecular weights of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Previously the method for determining protein molecular weights from SDS-PAGE depended on the accidental, only partial linearity of protein movement with the logarithm of its molecular weight. A new, mathematically rigorous method with supporting data is now described demonstrating that such movement is dependent upon the reciprocal of protein size. Experimental data, therefore, follow most closely a hyperbolic curve when plotted directly; it becomes linear and passes through the origin when movement is plotted vs the reciprocal of protein molecular weight. In the earlier method determination of the error of a measurement of molecular weight is very complex and never determined. In the method presented here such error is easily estimated and it is identical in both the hyperbolic and linear forms of data presentation. This method may eventually also allow other less-significant forces controlling movement such as protein charge to be analyzed and understood.     

An improved method for separation of low-molecular-weight polypeptides by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel

An improved system for SDS-polyacrylamide gel electrophoresis, capable of analyzing polypeptides having molecular weights from 1500 to 100,000 (especially showing high resolving power in the 1500 to 25,000 molecular weight range) is described. The 10 to 18% linear gradient gel containing 7 M urea with an acrylamide:bisacrylamide ratio of 20:1 and the Laemmli discontinuous buffer was used. The use of the gel with a high crosslinkage ratio is shown to be effective in lowering the leakage of low-molecular-weight polypeptides from the gel. This method has facilitated rapid detection of small amounts of low-molecular-weight polypeptides in body fluids by the use of silver stain. A procedure is presented for the elimination of false bands on the gel frequently encountered during silver staining. The separation patterns of enzymatic cleavage products of proteins, uremic plasma, and urines from nephropathy patients are illustrated. This system is also applicable in the separation of lipopolysaccharides and also for the detection of phospholipids.     

Identification of macromolecules by quantitative polyacrylamide gel electrophoresis and isoelectric focusing: a comparison of three approaches.

Identification of macromolecules by zone electrophoresis has usually been based on differences in migration distances under a single set of electrophoretic conditions. Classically, it has taken the form of coelectrophoresis on gel slabs. In “quantitative” polyacrylamide gel electrophoresis (PAGE), separation conditions were standardized sufficiently to allow for identification of macromolecules between experiments on the basis of their relative electrophoretic mobilities, R_f ± σ_Rf. More reliably, molecular identity or distinguishability have been based on several R_f values at several gel concentrations (%T) and the linear relationship between log R_f and %T, the Ferguson plot. The slope (retardation coefficient K_R of this plot is desoriptive of molecular size while the γ-intercept (Y₀) is a measure of net charge. The joint 95% confidence envelopes for K_R and Y₀ may be used as criteria for identification of molecules. Distinction between two molecular species depends on the size and position of the two confidence envelopes or ellipses. By pooling estimates of residual varlance (scatter areund the regression line for the Ferguson plot) for several proteins, it is possible to reduce the size of the ellipses and improve the sensitivity of the method to distinguish elesely related species. The sensitivity of this method depends on the size and reprodueibility of the 95% confidence envelopes, and on the limitatiens in the number of electrophoretic fractionations that one is reasonably willing to invest. Any molecular identification problem therefore raises the implieit question whether to base distinction on migration distance, on R_f, or on the joint 95% confidence envelopes for K_R and Y₀ and related statistical (F test) eriteria. Further, in the event of inconsistent answers to the question of molecular distinguishability from the three approaches, we need rational criteria to select the “best” answer. These problems and some solutions are illustrated by the present study which was designed to determine whether the enzymatic digestion products of human growth hormone produced by subtilisin-B are or are not the same as those obtained by digestion with plasmin. It appears that the joint 95% confidence envelopes of K_R and Y₀ provide at this time the most discriminating criteria of distinction, indicating significant differences between nearly all the products of plasmin and subtilisin digestion of hGH. However, the lower resolution provided by the R_f criteria has the advantage that it allows one to group the products of the partial hydrolysis of hGH into “families” which may be associated with different ranges of specific bioactivities.     

Human cytomegalovirus-induced immediate early antigens: analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after immunoprecipitation.

Immediate early antigen (IEA) induced in human lung fibroblasts by human cytomegalovirus was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation with IEA-positive human sera. Two polypeptides of 76,000 daltons (76K) and 82K were detectable within 90 min after infection. Polypeptides of similar molecular weight were also found in immunoprecipitates of human cytomegalovirus-infected cells nonpermissive for virus replication. IEA is located within the nucleus, although some of the 76K material appears to be located on the outer nuclear membrane. Raising salt concentrations in the extraction buffer increased antigen extraction. The contribution of these IEA polypeptides to IEA nuclear fluorescent staining is discussed.     

Activity staining of nucleolytic enzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Use of aqueous isopropanol to remove detergent from gels

The sensitivity with which RNase and DNase activity can be detected after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) varies widely, depending upon the particular SDS preparation used for electrophoresis. (See also [10.], Anal. Biochem. 100, 357–363.) Sensitivity of detection is greatly increased by using buffered 25% isopropanol, rather than buffer alone, to wash detergent from gels after electrophoresis. Thus it is routinely possible to detect bovine pancreatic RNase A at the picogram level. Use of isopropanol improved activity staining of RNases with each of the 10 SDS preparations examined, including one containing 32% tetradecyl sulfate and 4% hexadecyl sulfate, and reduced the variability from preparation to preparation observed when buffer alone was used to remove SDS. Other water-organic cosolvent binary mixtures can be used but none shows advantages over aqueous isopropanol when sensitivity of detection as well as availability and cost of organic solvent are considered.     

Transferrin variants in sheep: separation and characterization by polyacrylamide gel electrophoresis and isoelectric focusing*

Summary. Isoelectric focusing with carrier ampholytes in ultrathin polyacrylamide gels and polyacrylamide gel electrophoresis in a discontinuous buffer system were used for the separation of sheep transferrin variants. For identification of the different iron-binding sites of transferrin a stepwise urea gradient, different degrees of iron saturation and double one-dimensional electrophoresis were used. Isoelectric focusing results in an increased resolution of the Fe₀-transferrin, Fe₁-transferrin and Fe₂-transferrin region. At the level of Fe₀-transferrin and Fe₁-transferrin the variants I, A, G, B, C, D, M, E, Q, P can be identified. The method is especially suitable for genetic studies. For screening purposes up to 108 samples can be separated within one run in an ultrathin gel.     

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation–emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.     

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.     

Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels

We present a new method for visualizing proteins electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are incubated in CuCl2 to produce a negative image of colorless protein bands against a semiopaque background. Gels are stained completely within 5 min, do not require destaining, and can be stored indefinitely without loss of the image. Because proteins are not permanently fixed within the gel, they can be quantitatively eluted after chelation of Cu with EDTA. The sensitivity of the CuCl2 stain falls between that of Coomassie blue and silver. We anticipate that CuCl2 will be useful in the rapid analysis of proteins by polyacrylamide gel electrophoresis and in the preparation of purified polypeptides by elution from gel slices.