Fluorography of tritium-labelled proteins in immunoelectrophoresis

A method is described for detecting 3H-labelled proteins in immunoelectrophoretic systems performed on agarose gels. The method is based on the incorporation of a polyacrylamide gel into the agarose gel after the electrophoresis. This mixed gel has the characteristics of a polyacrylamide gel, making it possible to use fluorography as has been described for polyacrylamide gels. The applicability of the fluorography method is demonstrated by analyzing 3H-labelled human serum albumin and 3H-labelled pig intestinal brush border proteins by quantitative immunoelectrophoresis.     

Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots

We have determined that sodium salicylate, a water-soluble fluor which we use routinely for fluorography with polyacrylamide gels, is also useful for fluorography with thin-layer media. Detection of 3H-labeled material applied to thin-layer chromatography plates, or nitrocellulose membranes, can be enhanced up to 150-fold after treatment with an aqueous solution of 2 M sodium salicylate, while detection of 35S-labeled material is enhanced only about 2-fold. We demonstrate the utility of sodium salicylate fluorography in detecting 3H-labeled palmitic acid following thin-layer chromatography and 3H-labeled proteins following blotting to nitrocellulose.     

Localization and direct quantitation of 3H-labeled proteins and RNAs in slab gels by a new detection system

The distribution patterns of ³H-labeled proteins and RNAs in dehydrated polyacrylamide and agarose gels have been recorded with a newly developed instrument: the Linear Analyzer which has a position-sensitive detector that counts a whole gel lane simultaneously. The high sensitivity makes the Linear Analyzer a suitable instrument for direct quantitation of tritium labeled macromolecules in gels without pretreatment by impregnating reagents. The very low detection limit of the Linear Analyzer reduces substantially the time of evaluation in comparison to the relativly long exposure times needed in fluorography. The resolution of radioactivity profiles monitored by the Linear Analyzer reaches the quality attained by fluorography.     

Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography.

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.     

Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels

The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration.     

Fluorographic detection of radioactivity in polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its comparison with existing procedures.

A fluorographic procedure was optimized which utilized acetic acid as the solvent for 2,5-diphenyloxazole (PPO). This procedure was then compared with existing fluorographic procedures which utilize PPO in dimethyl sulphoxide or sodium salicylate in water, and a commercially available fluorographic solution. En3Hance (New England Nuclear Corp.). A comparison of the four methods revealed that all of the procedures resulted in essentially the same efficiency of radioactivity detection. The acetic acid/PPO procedure was found to have several technical advantages. There is no need to pre-fix proteins in gels, and either agarose or acrylamide gels can be used. The acetic acid/PPO procedure was found to be a simple, sensitive and efficient alternative fluorographic method.     

A simple and reliable method for the drying of polyacrylamide slab gels

A simple method for the drying of polyacrylamide slab gels is described. 2-mm thick gels with gradients of 5–20% acrylamide dry without complications. The dried gels are transparent permitting transmission densitometry and fluorography.     

DNA Restriction Fragment Length Polymorphism in Races of the Soybean Cyst Nematode,Heterodera glycines

Restriction endonuclease digests of total DNA from races 3, 4, and 5 of the soybean cyst nematode, Heterodera glycines, have been analyzed on agarose gels. DNA fragment patterns of race 4 were completely different from those patterns obtained for races 3 and 5 by all eight restriction enzymes tested. Differences in long and short restriction DNA fragments generated by the enzyme Msp I or its isoschizomer, Hpa II, were detected between race 3 and 5 digestion profiles. Rapid DNA isolation followed by its digestion with either Msp I or Hpa II enzymes and visualization of repetitive DNA fragments in agarose gels provided a diagnostic assay for the populations of the three races examined in this study.     

A technique for electrophoresis in multiple-concentration agarose gels

A technique has been developed for embedding several agarose gels (running gels), each of a different agarose concentration, within a single 1.5% agarose slab. Equal portions of a sample were placed at the origin of each running gel and were simultaneously subjected to electrophoresis. Protein within the running gels was detected by staining with Coomassie blue; 0.2% gels were the least concentrated gels that were stained without gel breakage. Using the above technique, the dependence of electrophoretic mobility on agarose concentration has been measured for bacteriophage T7 capsids and a capsid dimer.     

A versatile technique for drying polyacrylamide slab gels

Slab gels of different thickness, and of various homogeneous or gradient concentrations of acrylamide, can be dried rapidly and consistently using a newly developed drying procedure. After drying, stained gels retain excellent resolution of electrophoretic patterns, and unstained gels are suitable for use in autoradiography and fluorography.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Polyacrylamide lamination enables mass spectrometry compatible staining and in-gel digestion of proteins separated by agarose IEF

Agarose IEF enables the separation of large proteins and protein complexes. A complication of agarose gels attached onto polyester support is the lack of sensitive protein staining methods compatible with protein analysis and identification protocols. In this study, agarose IEF gels were used to separate the proteins, followed by layering the agarose with polyacrylamide. The formed laminate gels were seamless and durable and they were readily detached from the polyester. The gels were amenable to MS compatible staining. The sensitivity obtained with the acidic silver staining method was 20-50 ng/band of myoglobin. Laminated agarose was a suitable matrix for in-gel digestion based generation of tryptic peptides for MALDI-MS.     

Transient electric birefringence of agarose gels. II. Reversing electric fields and comparison with other polymer gels

The transient electric birefringence of low electroendosmosis (LE) agarose gels oriented by pulsed unidirectional electric fields was described in detail in Part I [J. Stellwagen and N. C. Stellwagen (1994), Biopolymers, Vol. 34, p. 187]. Here, the birefringence of LE agarose gels in rapidly reversing electric fields, similar in amplitude and duration to those used for field inversion gel electrophoresis, is reported. Symmetric reversing electric fields cause the sign of the birefringence of LE agarose gels, and hence the direction of orientation of the agarose fibers, to Oscillate in phase with the applied electric field. Because of long-lasting memory effects, the alternating sign of the birefringence appears to be due to metastable changes in gel structure induced by the electric field. If the reversing field pulses are equal in amplitude but different in duration, the orientation behavior depends critically on the applied voltage. If E < 7 V/cm, the amplitude of the birefringence gradually decreases with increasing pulse number and becomes unmeasurably small. However, if E > 7 V/cm, the amplitude of the birefringence increase more than 10-fold after ～ 20 pulses have been applied to the gel, suggesting that a cooperative change in gel structure has occurred. Because there is no concomitant change birefringence must be due to an increase in the number of agarose fibers and /or fiber bundles orienting in the lectric field, which in turn indicates a cooperatice breakdown of the noncovalent “junction zones” that corss-link the fibers in to the fgel matrix. The sign of the birefringence of LE agarose gels is always positive after extensive junction zone breakdown, indicating that the agarose fibers and fiber bundles preferentially orient parallel to the lectric field when they are freed from the constraints of the gel matrix. Three other gel-forming polymers, high electroendosmosis (HEEO) agarose (a more highly changed agarose), β-carrageenan (a stereoisomer of agarose), and polyacrylamide (a chemically corss-linked polymer) were alos studied in unidirectional and rapidly reversing electric fields. The birefringence of HEEO agarose backbone chain. The β-carrageenan gels exhibit variable orientation behavior in reversing electric fields, suggesting that its internal gel structure is not as tightly interconnected as that of agaroise gels. Both HEEO agarose and β-carrageenan gels exhibit a large increase in the amplitude of the birefringence with increasing pulse number when asymmetric reversing pulses > 7 V/cm are applied to the gels, suggesting that junction zone breakdown in a common feature of polysaccharide gels. Chemically cross-linked polyacrylamide gels exhibit very small birefringence signals, indicating that very little orientation occurs in pulsed lectric fields. The sign of the birefringence is independent of the polarity of the lectric field, as expected from the Kerr law, and normal orientation behavior is observed in reversing electric fields. Hence, the anomalous change in sign of the birefringence observed for agarose gels in reversing electric fields must be due to the metastable junction zones in the agarose gel matrix, which allow gel fiber rearrangements to occur. © 1994 John Wiley & Sons, Inc.     

Affinity electrophoresis: New simple and general methods of preparation of affinity gels

Two simple and generally applicable methods of preparation of affinity gels for affinity electrophoresis in agarose and polyacrylamide gels are described. In the first method, amino ligands are coupled to periodate-oxidized agarose gel beads (Sepharose 4B), and homogeneous affinity gels are obtained after mixing the melted substituted beads with either melted agarose solution or with the polymerization mixture used for the preparation of polyacrylamide gels. This type of affinity gel was used for affinity electrophoresis of lectins (immobilized p-aminophenyl glycosides), ribonuclease (immobilized uridine 3′,5′-diphosphate 5′-p-aminophenyl ester), trypsin (immobilized p-aminobenzamidine), and double-stranded phage DNA fragments (immobilized acriflavine). Alternatively, heterogeneous affinity gels are prepared from the suspension of ligand-substituted agarose, dextran, or polyacrylamide gel beads in the polymerization solution normally used for preparation of polyacrylamide electrophoretic gels. This technique was used for affinity electrophoresis of lectins, ribonuclease, and trypsin on affinity gels containing appropriate ligands coupled to the gel beads “activated” by various methods. Applicability of affinity gels prepared by the two methods described above for affinity isoelectric focusing is demonstrated.     

Preparative isoelectric focusing in agarose

The use of agarose gels as supporting media for flat-bed preparative isoelectric focusing was applied to the fractionation of serum proteins in the pH range 3.5–6, and red cell hemolysates in the pH range 3–8. The agarose gels are easy to prepare, give linear pH gradients, and do not appear to produce molecular sieving effects. Up to 1 g serum proteins can be loaded on the gels, with recoveries between 68 and 82%. Nucleoside phosphorylase from red cell lysates was recovered with 76% yield, indicating that no appreciable denaturation of this enzyme had occurred. Preparative isoelectric focusing in agarose gels provides a useful alternative to existing techniques of preparative isoelectric focusing in sucrose gradients or granulated gels.     

Stereocontrolled Synthesis of an Octasaccharide Part of I-Active Glycolipids

The effects of glycerin and ethylene glycol on the elastic modulus and DSC thermograms of agarose and kappa-carrageenan gels were examined to clarify the relation between structure and properties. The elastic modulus of these gels as a function of the concentration of polyols increased up to a certain concentration and then decreased with increasing concentration of polyols. These polyols shifted the melting temperature of the gel to higher temperatures in kappa-carrageenan gels but to lower temperatures in agarose gels. The temperature dependence of elastic modulus was changed in opposite directions in agarose and kappa-carrageenan gels by the addition of polyols, and this is discussed on the basis of model consisting of junction zones which are connected by Langevin chains. It was suggested that the mean distance between junction zones became shorter in the presence of a small amount of polyols.     

An affinity gel for the inhibition, binding, and isolation of serine proteases

A preparation procedure for gels for the specific binding and inhibition of serine proteases is described. Phosphoryl trifluoride was synthesized and reacted with two different types of agarose gels, a crosslinked agarose (Sepharose CL-4B) and an agarose containing spacer arms with terminal vicinal-diol groups (a hydrolyzed epoxy-activated Sepharose 6B). The phosphoryl difluoride groups coupled to the gels were, in both cases, further modified by treatment with isopropanol to obtain isopropyl fluorophosphate groups covalently bound to the matrix. It was found that both modified gels absorbed and inhibited plasmin, but that the modified gel with spacer arms was markedly more efficient.     

Rapid determination of chain length pattern in poly(ADP-ribose) samples

(³H)poly(ADP-ribose) synthesized from nuclei by incubation with (³H)NAD was released from protein by alkaline treatment and electrophoresed in dodecyl sulfate gels. Individual polymers up to at least 33 units were completely separated according to their chain length. Size distribution was visualized by fluorography of the gels, and quantified by radioactivity determination of sliced gels The method could be applied to crude nuclear extracts. It showed that nuclei of Ehrlich ascites tumor cells produced a poly(ADP-ribose) pattern distinctly different from that of rat liver nuclei.     

Electrophoresis, crossed immunoelectrophoresis, and isoelectric focusing in agarose gels with reduced electroendosmotic flow

An ion exchange method using QAE-Sephadex for preparation of agarose with a low electroendosmotic flow and reduced adsorption properties is described. The successful use of such agarose for the separation of highly cationic proteins is illustrated.A method for isoelectric focusing of proteins in gels made from a mixture of purified agarose and a water-soluble non-cross-linked acrylamide polymer is described. This method can be combined with immunochemical identification by electrophoresis of the separated components into antibody-containing agarose gels, also containing such a polymer of acrylamide.