Isoelectric focusing of proteins in rewetted ultrathin polyacrylamide gel layers

Ultrathin layers of polyacrylamide gel bound to glass can be washed, air-dried, and stored for at least 1 year before rewetting in ampholyte solutions for isoelectric focusing. Short-term drying affects neither fluorescent banding of the ampholytes (not evident in conventional gels) nor resolution of complex protein mixtures while prolonged storage seems to have no deleterious effects. Layers are fully functional after soaking for 10 min in solutions that may contain 8 M urea and 10% sorbitol. Rewetting allows the rapid survey of different ampholytes, gradient stabilizers, separator compounds, or protein reagents and is adaptable to concentration modification of the pH gradient (alone or with a gel overlay), to focusing in a transverse urea gradient, and to electrophoresis across a preformed pH gradient. The procedure avoids protein modification by residual polymerizing reagents while adding to the convenience and economy of using ultrathin layers in relatively small formats.     

Isoelectric focusing of multiple forms of DNase in thin layers of polyacrylamide gel and detection of enzymatic activity with a zymogram method following separation

Multiple forms of bovine pancreatic DNase (DNases A, B, C, and D) are separated by isoelectric focusing in thin layers of polyacrylamide gel with a carrier ampholyte in the pH range 4–6. The isoelectric points of DNases A, B, C, and D are 5.22, 4.96, 5.06, and 4.78, respectively. A zymogram method for detecting DNase activity as bands in the gel following isoelectric focusing is described. The method detects microgram amounts of DNase and has only one step. It can be used with the parified cazyme as well as with crude extracts of tissues containing DNase. By this method, two major components of DNase in ovine pancreas and at least three in malted barley as well as two previously unideatified forms of DNase in bovine pancreas with isoelectric points of 5.12 and 5.48 (DNases E and F) are observed.     

Characterization of alpha-1-antitrypsin by isoelectric focusing on an ultrathin polyacrylamide gel layer

Summary Through the use of ultrathin layer polyacrylamide gel isoelectric focusing it is possible to obtain a resolution of the bands of ₁AT so as to be able to easily recognize all six PiM subtypes. The optimal resolution of the PiM subtypes is obtained without deforming the pattern of the Pi phenotypes. In addition to high resolution, ultrathin layer polyacrylamide gel isoelectric focusing permits a notable reduction of fees to 1/5 of the usual.     

Two-dimensional polyacrylamide gel electrophoresis of the protease SP220K, a renal cell carcinoma marker

The present study analyses, by two-dimensional polyacrylamide gel electrophoresis, the protease SP220K isolated from renal cell carcinoma. The pure molecule is separated using either immobilized pH gradient or isoelectric focusing in conventional carrier ampholyte in the first dimension. Some interactions with the acrylamide matrix in isoelectric focusing are discussed. The results demonstrate that two-dimensional gel electrophoresis performed with enriched media such as basolateral membranes, allows the detection of the protease. In addition, the non detection of the molecule up to now by this methodology can be explained by the high tendency of oligomerization of SP220K. Effectively the high molecular weight form of the molecule of 220 kDa is favoured in two-dimensional gel electrophoresis over monomeric forms which are better detected in SDS PAGE. This was confirmed by immunostaining performed with an antiserum to SP220K produced by nitrocellulose-bound antigen.     

RAT BRAIN TUBULIN: SUBUNIT HETEROGENEITY AND PHOSPHORYLATION

Abstract— Evidence for multiple forms of the α and β subunits of tubulin isolated from rat brain has been obtained by means of SDS polyacrylamide gel electrophoresis, isoelectric focusing, and SDS hydroxylapatite column chromatography. Fourteen distinct bands, localized near pH 5.4, were formed when tubulin was subjected to isoelectric focusing in a gradient established with a very narrow range ampholyte mixture. Three tubulin subunits, a₁., α₂, and β, were separated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in a second dimension. The β subunit was more acidic than the α subunits. Brain sections were incubated in tissue culture medium containing ³²P₁ and radiolabeled tubulin was subsequently isolated and subjected to electrophoresis. Only the β subunit was labeled. All radioactivity was associated with two or three adjacent bands on isoelectric focusing gels.     

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equpiment: a two-peak transient in the approach-to-equilibrium.

Using a semiporous plug of agar gel to support a sucrose density gradient column without restricting electrical conductivity, Massey and Deal [J. Biol. Chem.248, 56 (1973)] were able to use a conventional polyacrylamide gel electrophoresis apparatus to carry out single tube isoelectric focusing experiments in density gradients in only 2 hr using minute amounts (50 μg) of sample and very little ampholyte (0.18 ml); no cooling apparatus was required. In this work we report that 1) polyacrylamide provides a superior gel plug and 2) that ten isoelectric focusing tubes can easily be run simultaneously in a conventional polyacrylamide gel electrophoresis apparatus. In addition, the isoelectric points of eight proteins, with pI values ranging from 5.1 to 8.8 have been determined and the kinetics of the approach-to-isoelectric-focusing-equilibrium have been analyzed. Of special interest is the discovery that in the initial stages of focusing, in these sucrose density gradients, a major peak is formed at each end of the column; these two peaks migrate toward each other and finally coalesce into a single peak. Similar, although less pronounced, effects were previously observed by Catsimpoolas and Wang [Anal. Biochem.39, 141 (1971)] in focusing experiments in polyacrylamide gels. With all other conditions constant, the time required to reach equilibrium is 1) less in broad range (e.g., 3–10) pH gradients than it is in narrow range (e.g., 5–8) pH gradients and 2) generally greater with higher molecular weight substances than with lower molecular weight substances. Explanations are given for all of these kinetic phenomena.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein-containing TaqMan probes

The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland–Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways—both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.     

Human alpha 1-antitrypsin genetic polymorphism: PI N subtypes

Three new genetic variants (PI types) of alpha 1-antitrypsin are described. They have been compared to previously described phenotypes by several techniques including narrow pH range isoelectric focusing in ultrathin polyacrylamide gels. In this system, the relevant alpha 1-antitrypsin gel bands, identified by crossed immunoelectrophoresis, focused between PI M2, the most cathodal PI M subtype, and PI P BUD, the most anodal PI P subtype. They were therefore considered to be PI N subtypes. Two of them, PI N GRO and PI N YER, could not be separated by isoelectric focusing, but gave a different pattern in agarose gel electrophoresis. None of the new alleles seemed to be associated with disease. The high resolving power of isoelectric focusing is emphasized with respect to the information it may provide concerning amino acid substitutions, while the use of other techniques proved to be of utmost importance in the differentiation of other variants showing similar isoelectric points.     

Prenyltransferase from Saccharomyces cerevisiae. Purification to homogeneity and molecular properties.

Prenyltransferase (EC 2.5.1.1) has been purified to homogeneity from the supernatant fraction of yeast by ammonium sulfate fractionation, diethylaminoethyl-cellulose and hydroxylapatite chromatography, and column isoelectric focusing techniques. The active enzyme from isoelectric focusing columns emerged as a single symmetrical peak with specific activities 15- to 35-fold higher than previously reported preparations. The enzyme was found to be homogeneous by continuous polyacrylamide gel electrophoresis at pH 8.4 and discontinuous polyacrylamide gel electrophoresis at pH 6.9 as well as sodium dodecyl sulfate polyacrylamide electrophoresis at pH 7.0. By means of gel chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was shown to be a dimer with a molecular weight of 84,000 plus or minus 10%. The isoelectric point of the enzyme was determined to be 5.3. The enzyme synthesizes farnesyl and geranylgeranyl pyrophosphates from dimethylallyl, geranyl, and farnesyl pyrophosphates. Michaelis constants for the enzyme were 4, 8, and 14 mu M for isopentenyl, dimethylallyl, and geranyl pyrophosphates, respectively.     

Poly-N-acryloyl-Tris gels as anticonvection media for electrophoresis and isoelectric focusing

We describe in detail the synthesis of an acrylic monomer, N-acryloyl-tris(hydroxy-methyl)aminomethane (NAT), which was successfully used for the preparation of gels for electrophoresis and isoelectric focusing. The polymerization kinetics and transparency of the poly(NAT) gels crosslinked by N,N'-methylenebisacrylamide (Bis) are also shown. Poly(NAT)-Bis gradient (4-24%) gel resolves proteins according to their size. The exclusion limit of this gel is slightly over 3 X 10(6), which is more than threefold higher than the exclusion limit of the polyacrylamide gradient gel of the same concentration. The gel made of 6% NAT and 3% Bis represents a suitable matrix for isoelectric focusing. These results demonstrate that poly(NAT)-Bis gels could be advantageously used in those applications where the extensive sieving by the polyacrylamide matrix is not desir desirable.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

Ultrathin-layer isoelectric focusing in polyacrylamide gels on cellophane.

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.     

Multiple molecular forms of cytochrome P-450SCC purified from bovine corpus luteum mitochondria

Cytochrome P-450 related to side-chain cleavage of cholesterol (P-450SCC) was isolated from bovine corpus luteum mitochondria in the form of its stable cholesterol complex. The isolation procedure included ammonium sulfate fractionation and chromatography on omega-aminohexyl-Sepharose (AH-Sepharose). Corpus luteum P-450SCC was resolved into one minor (AH-I) and two major (AH-II and AH-III) fractions by the chromatography. Results of re-chromatography suggested the possibility that AH-III Fraction was originally complexed with lipidic material. The two major fractions purified by the re-chromatography (AH-IIR and AH-IIIR Fractions) showed essentially a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and their absorption spectra were indistinguishable from each other. Both fractions were further resolved into two major and some minor bands of P-450SCC by isoelectric focusing on polyacrylamide gel in the presence of a non-ionic detergent, as detected by protein staining, heme staining and immunoblot analysis with anti-bovine P-450SCC monoclonal antibody. Both AH-IIR and AH-IIIR Fractions were further resolved by high-performance liquid chromatography (HPLC) on SP-TSK gel column into two fractions, SP-I and SP-II. These fractions had the same N-terminal amino acid sequence, showed similar catalytic activity and resolved into one major and a few minor bands on isoelectric focusing on polyacrylamide gel. Much more heterogeneity was observed in purified P-450SCC preparations from bovine adrenal cortex mitochondria. These results indicated the presence of multiple molecular forms of corpus luteum P-450SCC as well as adrenal cortex P-450SCC. Computer simulation studies were carried out in order to analyze the mechanism of formation of multiple bands on isoelectric focusing. The multiple bands of corpus luteum P-450SCC could be explained by postulating the presence of two isozymes (or molecular forms) having a pair of sites each with or without a charged group.     

Heterogeneity of human alpha-fetoprotein as revealed by isoelectric focusing in urea-containing gels.

The heterogeneity of human alpha-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human alpha-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major alpha-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of alpha-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain alpha-fetoprotein charge isomers in various alpha-fetoprotein isolates may be important in considering certain biological functions of this protein.     

Superoxide dismutase isoenzymes in bovine and human milk

The presence of superoxide dismutase in bovine and human milk was investigated by ultrafiltration, gel filtration, and isoelectric focusing. Conclusive evidence for the presence of this enzyme in both milks is presented. The molecular weight of the enzyme was estimated by gel filtration on Sephadex G-100 to be 30,000, which is consistent with reported values for the copper, zinc form of superoxide dismutase. In addition, enzyme activity was inhibited by cyanide, thus eliminating the possibility that the enzyme was present in the manganese form. Several isoenzymes were detected by isoelectric focusing in polyacrylamide gel, and the isoenzyme pattern in bovine milk was the same as that found for bovine plasma, suggesting that milk superoxide dismutase originates from plasma. It may be that the presence of copper, zinc superoxide dismutase in milk is important for the maintenance of its oxidative stability.     

Purification and properties of monomeric cytochrome f from charlock, Sinapis arvensis L

Monomeric cytochrome f has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, isoelectric focusing and analytical ultracentrifugation, from the leaves of charlock, Sinapis arvensis L. The cytochrome was obtained in an aqueous extract following extraction of leaf lipids with butan-2-one, and was subsequently purified by acetone precipitation and chromatography on DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The purified cytochrome had adsorbance ratios of A422/A280 = 7.3 and A554/A280 = 1.07 in the reduced form. There was no indication of the presence of an absorbance band at 695 nm in the oxidised form. The cytochrome had a midpoint redox potential of +365 mV and was oxidised very rapidly by parsley plastocyanin. The molecular weight of the cytochrome was approximately 27 000 as determined by sedimentation equilibrium and by polyacrylamide gel electrophoresis in the presence of dodecylsulphate. The sedimentation coefficient (so20,w) of cytochrome f was 2.48 S. The cytochrome had an isoelectric point at pH 5.50 determined by isoelectric focusing in polyacrylamide gels.     

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.     

Isoelectric focusing as the crow flies

The evolution of isoelectric focusing is traced back over the years, from a somewhat shaky origin to present-day immobilized pH gradients. Four generations of methodology are classified and discussed: (A) Kolin's approach, consisting of a two-step technique, generation of a pH gradient by diffusion followed by a rapid electrokinetic protein separation; (B) Svensson-Rilbe's approach, consisting of creating a pH gradient in an electric field by utilizing as buffers a multitude of carrier ampholytes, i.e. of amphoteric species possessing good buffering capacity and conductivity at their pI; (C) immobilized pH gradients, by which non-amphoteric buffers and titrants (acrylamido weak acids and bases), titrated around their pK values, are grafted (insolubilized) onto a polyacrylamide gel matrix and (D) mixed-bed carrier ampholyte-Immobiline gel, by which a soluble, carrier ampholyte generated pH gradient coexists in the same matrix with an insoluble, Immobiline generated, pH gradient.