Continuous Buffer Research Articles

Unusual Electrophoretic Properties of the Coat Protein of Raspberry Ringspot Nepovirus

Coat protein of raspberry ringspot nepovirus (RRV) migrated during electrophoresis in a discontinuous buffer system as two components which were capable of interchanging during this procedure. However, only one component was formed during electrophoresis in a continuous buffer system. Treatment of the protein before electrophoresis with iodoacetamide, which alkylates sulfhydryl groups, prevented the formation of the faster migrating electrophoretic component. Thus the faster migrating component is probably an artifact caused by S-S bond formation. Although the coat proteins of two other nepoviruses did not behave in this way, the results obtained with RRV emphasize the need for caution in interpreting electrophoretic heterogeneity in virus proteins. The estimated molecular weight of RRV coat protein was 57,000.

Program in BASIC for Ferguson plot analysis, using a personal computer: application to gel electrophoresis in a continuous buffer.

A program in BASIC suitable for personal computers is described which is applicable to gel electrophoresis conducted in a single (continuous) buffer. The curve fitting is to a polynomial function, allowing for an objective selection of the most appropriate curve type and order--linear, convex or concave--in the particular application. Results do not differ significantly from previous programs for evaluation of linear Ferguson plots or of curve fitting to an exponential function for evaluating convex plots, executed on mainframe computers such as the DEC-10 (Digital) and IBM 370 computers. Thus, the program combines original versatility with, for the first time, the possibility for widespread application of Ferguson plot analysis on personal computers.

Linear Ferguson plots of polystyrene sulfate size standards for the quantitative agarose gel electrophoresis of subcellular particles.

Accurately standardized commercial polystyrene sulfate particles in agarose gel electrophoresis yield linear Ferguson plots at pH 7.4 over a gel concentration range up to 0.9% agarose which do not exhibit any significant sigmoidal curve elements, using either a discontinuous buffer system or a continuous buffer. Ferguson plots of these standard-sized particles were evaluated using alternatively a linear or convex model, by means of a newly developed set of programs (to be used in conjunction with program M-LAB) which (i) is sufficiently user-friendly to allow for quantitative agarose gel electrophoresis of subcellular-sized spherical particles based on their convex Ferguson plots with the same operational simplicity previously available for linear Ferguson plots only; (ii) simultaneously and interactively analyzes the Ferguson plots of all particles under consideration on the basis of an extended Ogston model.

Physical identification of a virus in a crude leaf extract by its Ferguson plot in agarose gel electrophoresis.

Crude extracts of turnip crinkle virus upon agarose gel electrophoresis yield (i) virus patterns unperturbed by contaminants; (ii) plots of mobility vs. gel concentration (Ferguson plots) parallel with those of the purified virus. The parallelism suggests similarity in size and shape but a lower net charge for the crude virus. This result is obtained when gel electrophoresis is carried out either in a continuous buffer or in a discontinuous (moving boundary electrophoresis) buffer system. The latter mode has the substantial benefit of electrophoretic (auto-)concentration of dilute virus sample prior to resolution. Thus, the Ferguson plot analysis in a discontinuous buffer system of turnip crinkle virus can be viewed as a model procedure for the physical identification of other viruses contained in dilute extracts, feasible even in the absence of a prior knowledge as to the nature of, or isolation of, the virus.

Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients.

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.

Microvascular transport and endothelial cell alterations preceding skeletal muscle damage in ischemia and reperfusion injury

We determined the leakage of macromolecules using FITC-dextran-150 as a tracer and measured the extent of no-reflow phenomenon by video field analysis. The cremaster muscle of anesthetized rats was fashioned as a single layer, splayed on a lucite chamber and suffused with bicarbonate solution at 35 °C. After a 1 hour period of baseline data collection, ischemia was produced by cross-clamping the cremasteric vascular pedicle for periods of 30 minutes and 2 hours in separate experiments. Macromolecular leakage was visualized after reinstitution of perfusion. Leakage occurred at postcapillary venules 15 to 50 μm in diameter and quickly spread to the interstitium. The magnitude of leakage decreased as a function of time with continuous buffer suffusion, but remained higher than in the control period. No reflow occurred in approximately 30 percent of the muscle microvasculature upon reperfusion. The no-reflow values at 30 minute and 2 hour periods of ischemia were significantly different from the control values but were not from each other. Electron micrographs demonstrated endothelial cell swelling and migration of leukocytes and normal myocytes after 1 hour of reperfusion following 2 hours of ischemia. Our results demonstrate that permeability changes, occurrence of no reflow, and leukocyte migration precede the onset of damage to skeletal muscle in ischemia and reperfusion injury.

Moving boundary electrophoresis on agarose gel of plant viruses and polystyrene microspheres

AbstractThree plant viruses: turnip crinkle (TCV), hibiscus chlorotic ringspot (HCRSV) and pelargonium flowerbreak (PFBV), and polystyrene size standards with radii of 22.4–59.4 nm can be stacked within trailing and leading ion net mobilities of 0.059 to 0.273 (relative to Na+). Stacking was carried out at pH 6.50, 0.03 M ionic strength, 50 mM 3–[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propane sulfonate, at a gel concentration of 0.4 %, in agarose gel electrophoresis conducted at 1.2 mA/cm2 of gel. Unstacking occurs under the same conditions at gel concentrations ranging from 0.5 to 1.1 % agarose, while it can be brought about between 0.1 and 0.7 % agarose when the pH is raised to 7.27, corresponding to a front moving boundary with a trailing ion net mobility of 0.216 (relative to Na+). Ferguson plots of viruses and polystyrene particles in the discontinuous buffer system are curvilinear and comparable to those obtained in a continuous buffer at pH 6.50 of the same composition and operative pH as that of the resolving phase of the discontinuous buffer. Particle radii and net charge values can be obtained from the non‐linear Ferguson plot in the discontinuous buffer system by previously reported methods of computer simulation, but this Ferguson plot presents a more limited data base than that in the continuous buffer since it excludes gel concentrations which yield relative mobility (Rf) values of 1.0. Since computer simulation provides the range of gel concentrations in which both the fiber radius and length [1], as well as the size of the particle [2], remain constant for a particular preparation of agarose, a simplified alternative method of evaluating particle sizes exists. Within that specific gel concentration range, the linear segment of the Ferguson plot can be used to compute particle sizes by an operationally convenient, albeit approximative, method, using the same PAGE‐PACK programs of D. Rodbard which are commonly used in the size determination of macromolecules by polyacrylamide gel electrophoresis. The two methods of particle size determination, based on either the entire non‐linear Ferguson plot or on its linear segment in the appropriate gel concentration range, yield similar results (average deviation 12 %). The radii of three plant viruses are dependent to different degrees on the presence of Ca++ in the electrophoretic system. Values obtained in the presence of Ca++ are comparable to those found by electron microscopy.

Detection of In Vitro and In Vivo Cleavage of High Molecular Weight Kininogen in Human Plasma by Immunoblotting With Monoclonal Antibodies

Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti-light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

Detection of in vitro and in vivo cleavage of high molecular weight kininogen in human plasma by immunoblotting with monoclonal antibodies

Purified human high-mol-wt kininogen (HMWK), the cofactor of the contact phase of blood coagulation, migrated as a single band (approximately 110,000 mol wt) in a continuous buffer sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), but appeared as two separated bands (approximately 120,000 and 105,000 mol wt) when analyzed in a discontinuous buffer SDS-PAGE system. After elution from SDS polyacrylamide gels, each of the two bands showed coagulant activity. Six murine monoclonal antibodies (Mabs) against HMWK were produced and purified. In immunoblotting studies, three Mabs bound to the isolated alkylated heavy chain and one to the alkylated light chain of HMWK, whereas the remaining two bound only to the single-chain or unreduced two-chain molecule. None of the Mabs inhibited the clotting activity of HMWK or its binding to kaolin. Two of the Mabs, one directed against the light chain and one against the heavy chain, were used as specific probes to study HMWK in plasma samples using an immunoblotting technique. The anti-light chain Mab identified two distinct bands (approximately 120,000 and approximately 105,000 mol wt) in normal human plasma, but not in plasma from patients with hereditary HMWK deficiency. The anti-heavy chain Mab detected two additional bands (approximately 60,000 and approximately 54,000 mol wt) corresponding to low-mol-wt kininogen (LMWK) in normal plasma. A sensitive and specific quantitative immunoblotting assay of HMWK antigen in plasma was developed. Moreover, the immunoblotting technique with the anti-light chain Mab was used to detect the cleavage of HMWK in plasma samples after in vitro or in vivo activation of the contact system. The anti- light chain Mab demonstrated in vivo activation and cleavage of HMWK during an angioedema attack in a patient with hereditary angioedema and C1-inhibitor deficiency.

Binding of lithium dodecyl sulfate to polyacrylamide gel at 4°C perturbs electrophoresis of proteins

Although polyacrylamide gel has no affinity to lithium dodecyl sulfate (LDS) at 25°C, the gel maximally binds 17 mg of LDS per gram dry weight at 4°C. When polyacrylamide gel electrophoresis is carried out at 4°C in the presence of LDS instead of sodium dodecyl sulfate (SDS) using a continuous buffer system, migration of proteins with lower molecular weight is accelerated as a result of the deficiency of LDS in the frontal region of the gel. When the gel is saturated with LDS, electrophoresis in the presence of LDS at 4°C shows a resolution higher than that of SDS-polyacrylamide gel electrophoresis at 25°C.

Simple polycrylamide gel electrophoresis in continuous carbonate buffer system suitable for the analysis of ascitic fluids of hybridoma bearing mice

A convenient simple polyacrylamide gel electrophoresis system is described, suitable for semiquantitative analysis of the content of monoclonal antibodies in ascitic fluids of hybridoma-bearing mice.

A device for preparative elution electrophoresis using a polyacrylamide gel slab

AbstractA compact device, capable of being clipped onto the end of a 3 mm thick analytical polyacrylamide slab gel, has been developed for the collection of resolved protein zones as they elute from the end of the gel. Its unique feature is that eluted proteins were collected between two polyacrylamide‐embedded paper membranes and removed either by continuous or intermittent buffer elution. Because of this direct coupling to large gels identical to those used for analytical electrophoresis, protein zones were obtained with high resolution. The method was applicable to non‐denaturing cathodic and anodic electrophoresis, as well as sodium dodecyl sulfate electrophoresis. Medium‐scale quantities (up to 10 mg/cm2 cross‐sectional area) of protein were fractionated in as little as 4 h with high recoveries.

Structure of covalent insulin-receptor complexes (I-S-S-R) in isolated rat adipocytes and human placental membranes.

The structure of naturally-formed covalent disulphide-linked complexes between insulin and its receptor was examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. To prevent destabilization of disulphide bonds at alkaline pH the standard discontinuous electrophoresis conditions were changed to a continuous buffer system at pH 7.0. 125I-insulin was first bound to either rat adipocytes or human placental membranes for 10 min at 37 degrees C. After washing, non-dissociable radioactivity was extracted from cells or membranes in Triton X-100 and immunoprecipitated with an antiserum (B-2) to the insulin receptor. Electrophoresis of the immune precipitate revealed the two smaller of the three reported species of native insulin receptor (Mr values approx. 350 000, 290 000 and 260 000); in addition, a species of Mr 200 000 was also frequently observed in adipocytes. When non-dissociable 125I-insulin was chemically crosslinked to adipocytes or placental membranes, prior to solubilization and immunoprecipitation, all three species of the native receptor were labelled; after reduction, only a single species of Mr 130000 was observed. These findings indicate that disulphide exchange of insulin occurs with the Mr 130000 (alpha) binding subunit within partially reduced species of the native, oligomeric receptor. The degree of disulphide binding of insulin could therefore depend on the relative abundance of partially reduced receptor species and on the redox state of the cell membrane.

Electrophoretic analysis of the estrogen receptor. Molybdate stabilization and identification of the classical estrogen receptor.

Conditions are defined which permit analysis of estrogen receptors from the mammalian uterus by polyacrylamide gel electrophoresis, thereby solving a longstanding problem encountered in previous attempts at such analysis, namely the failure of a large portion of the receptor population to enter such gels. A paramount requirement for entry of the estrogen-receptor complex into polyacrylamide gels is its maintenance in an untransformed state which does not form aggregates that are excluded from these gels. Of the multiple estrogen-binding proteins separated, only one (relative mobility of 0.5-0.6) possessed the definitive characteristics of the classical estrogen receptor. The inclusion of molybdate in extraction buffers selectively enhanced receptor recovery and facilitated its separation. Moreover, the estrogen-receptor complex so resolved is separated from other types of estrogen-binding proteins present in the uterine cytosol. These findings show that the molybdate-stabilized estrogen receptor exists in a single discrete form, but otherwise exhibits multiple forms that are probably artifactual. Electrophoresis in discontinuous buffers, but not in a continuous buffer system, promoted aggregate formation. This finding has implications concerning the subunit structure of the untransformed receptor.

Esterase D: Simultaneous electrophoretic determination of the three common allozymes (ESD 1, 2, 5)

AbstractA simple and time‐saving electrophoretic method for the determination of the 6 common phenotypes of red cell esterase D (ESD) is presented. Using a continuous buffer system composed of malic acid, the separation of the allozymes is accomplished at pH‐values between 5.35–5.45 by thin‐layer agarose gel electrophoresis in 4 h. Until now, a combination of electrophoresis and isoelectric focusing was required to obtain the same results. The economical advantage as well as the modest requirements concerning laboratory equipment are evident.

Electrophoresis buffers for polyacrylamide gels at various pH

Continuous electrophoresis buffers are described for polyacrylamide gels at pH values ranging from 3.8 to 10.2. The buffers consist of an acidic and a basic component with p K values near the pH of the buffer. The pH is maintained to within 0.5 pH unit in the electrode compartments during prolonged electrophoresis. Some proteins produce clear bands on gels with each of the 10 buffers. The buffers provide an expansion of the pH range of gel electrophoresis and are likely to be useful in the detection of genetic variation in proteins and in other applications.

Assessment of Protein Digestibility by In Vitro Enzymatic Hydrolysis with Simultaneous Dialysis

An in vitro digestion method to assess the quality of proteins based on enzymatic hydrolysis is presented. The method consists of a two-step proteolysis at 37°, a 30-minute incubation of the protein with pepsin at pH 1.9, followed by pancreatin digestion at pH 8 for 24 hours in a dialysis bag with a 1000 molecular weight cutoff for the continuous elimination of the digested products into a replaceable buffer. Three types of buffer replacement were tried. These were, in increasing order of efficiency: intermittent with either six changes (infrequent buffer replacement) or eleven changes (frequent buffer replacement) or continuously circulated at the rate of 212 ml/hour (continuous buffer replacement). By using three protein sources, i.e., casein, soybean and rapeseed proteins, it was found that the degree of digestion (dialyzed N) as well as the regularity of the process were markedly improved by increasing the frequency of buffer replacement. In spite of different pepsin digestibilities, as determined from the production of trichloroacetic acid-soluble N, the digestion of the three proteins gave equivalent results when the best procedure of buffer replacement was used (continuous buffer replacement). The deleterious effect of heat and alkali treatment on protein digestion was readily shown by this procedure and indicated casein to be a very heat-sensitive protein as compared to the others.protein digestibility enzymatic hydroxlysis dialysis pepsin pancreatin

Characterization of vaccinia polypeptides

Structural proteins of vaccinia virus (IHD-J strain) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis. Glycoproteins and phosphoproteins were identified. Correlations between the bands revealed by the continuous buffer method and those developed by the discontinuous buffer method were confirmed by two-dimensional SDS-PAGE, and the spots in the two-dimensional electrophoresis (first, isoelectrofocusing; second, SDS-PAGE) were identified by parallel electrophoresis of selectively solubilized polypeptides. Double labeling of the proteins with [ 3H]fucose and [ 14C]glucosamine showed that the proteins of virus grown in KB cells were glycosylated. When the virus was grown in L929 cells, the carbohydrate moieties lacked fucose, while glucosamine was detected in the same 14 polypeptides as in virus grown in KB cells. All glycopolypeptides migrated as clusters of spots upon two-dimensional electrophoresis. 32P incorporation was found in nine bands, five of which coincided with [ 35Szmethionine-labeled proteins. The phosphorylated polypeptides also migrated as multiple spots upon two-dimensional electrophoresis. The results indicated that the structural polypeptides of vaccinia virus detected as 39 bands in one-dimensional gels and 84 spots in two-dimensional profiles could be assigned to 52 polypeptide species, of which about 50% were basic.

Phytohemagglutinin isolectin subunit composition

The subunit compositions of individual phytohemagglutinin isolectins from red kidney bean Phaseolus vulgaris were examined by isoelectric focusing and sodium dodecyl sulfate electrophoresis on polyacrylamide gels. Isoelectric focusing reveals heterogeneous but unique and non-overlapping protein band patterns for each of the homotetrameric isolectins, E 4 and L 4. Isoelectric focusing of the intermediate isolectins which contain both subunits (E 3L 1, E 2L 2, and E 1L 3) show all the protein bands common to isolectins E 4 or L 4 in proportions relative to their suggested subunit compositions. Polyacrylamide gel electrophoresis in a continuous sodium dodecyl sulfate buffer system gives a single protein band for all of the isolectins. In contrast, a discontinuous sodium dodecyl sulfate buffer procedure resolves isolectins E 4 and L 4 into single major protein bands of apparent molecular weights 31 700 (±600) and 29 900 (±200), respectively. Each of the intermediate isolectins contained both protein bands and their relative proportion, as determined by absorbance scanning, confirms the phytohemagglutinin isolectin subunit compositions as E 4, E 3L 1, E 2L 2, E 1L 3, and L 4.

Electrophoresis in gels containing zwitterionic groups

AbstractA zwitterionic methacrylamide derivative, (3‐sulfopropyl)dimethy1(3‐methacrylamidopropyl) ammonium inner salt (MAPS), was synthesized and then copolymerized with N,N′‐methylenebisacrylamide (Bis) to make gels carrying covalently bound sulfobetaine groups. Gels were also prepared with the addition of (3‐sulfopropyl)trimethyl ammonium inner salt (TMAPS) to Bis‐crosslinked polyacrylamide prior to polymerization. The properties of these gels in gel electrophoresis and electrofocusing were tested and compared with those of Bis‐crosslinked polyacrylamide.In MAPS gels the absolute migration velocities of proteins and dyes at either polarity of migration were decreased. Anionic species were much more strongly retarded than cationic species. Ferguson plots (based on protein mobilities relative to dye in a continuous buffer) of catalase at pH 4 obtained on MAPS gels had a similar slope but lower y‐intercept compared with those on equivalent polyacrylamide gels, indicating that the available (effective) net charge on the protein was decreased because it interacts with the zwitterionic charge on the gel. Similarly, the addition of TMAPS to polyacrylamide decreased the relative catalase mobility at non‐restrictive gel concentrations. However, at gel concentrations above 4 %T, relative mobilities were increased, presumably because the effective pore size of the gel was increased through inhibition of polymerization by the zwitterionic compound.MAPS gels did not exhibit electroendosmosis by the criterion of cyanocobalamin displacement. Resolution between peptides in MAPS gels, and between proteins in ternary copolymer gels made of MAPS, Bis and polyacrylamide, was similar to that on polyacrylamide. Bands of catalase appeared sharpened in MAPS gels. pH Gradients form, decay and exhibit conductance gaps in MAPS electrofocusing gels and in polyacrylamide gels in presence of 0.25‐1.5 M TMAPS, in a manner which was qualitatively indistinguishable from polyacrylamide.