The effect of three variables on adsorption of rabbit IgG to colloidal gold

The purpose of this investigation was to (a) begin to evaluate the coagulation curve as a means of selecting the minimal quantity of IgG required to stabilize colloidal gold (Au); (b) determine the effect of the quantity of IgG added, the pH of adsorption, and the isoelectric point (pI) range of the IgG on the quantity of IgG bound and the stability of the IgG-Au complex with respect to desorption; and (c) discuss these results with respect to current theory on the effect of pH on adsorption of IgG to surfaces. No absolute minimal value required to prevent coagulation could be determined despite the high reproducibility of the values obtained; approximate values were selected. Each variable had an effect on the quantity of IgG bound: as the quantity of IgG added increased, the quantity bound increased; as the pH of adsorption became more alkaline, the quantity bound decreased; and as the pI range of the IgG became more alkaline, the quantity bound increased. IgG-Au complexes with a variable number of bound IgG molecules, depending on the three variables selected, can be produced. Production of IgG-Au composed of uniform numbers of IgG is discussed. A modification of the current theory on the effect of pH on adsorption of IgG is proposed.     

The effect of immune complexes on the electrophoretic mobility of sheep erythrocytes

Influence of concentration of aggregated immunoglobulins with varying molecular weights on the electrophoretic mobility of ram erythrocytes has been explored. It was shown, that electrophoretic mobility of ram erythrocytes depend on the quantity and the size of adsorption complexes.     

Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this protocol, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided.     

Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions

Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5–10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.     

A method for rapid quantitation and preparation of antithrombin III-high-affinity heparin fractions

An electrophoretic method for the quantitation and preparation of antithrombin III-high-affinity heparin using agarose beds is described. The method allows the determination of high-affinity heparin fractions in several samples in one single step. The incubation mixture containing heparin and antithrombin III is submitted to agarose gel electrophoresis in 0.06 m barbital buffer, pH 8.6. A sharp separation between free antithrombin III, the complex antithrombin III-heparin, and free heparin occurs under these conditions. Around 30% of heparin molecules present in commerical preparations bind to antithrombin. This bound heparin has an anticoagulant activity of 240 IU. Negligible binding of other sulfated mucopolysaccharides to antithrombin III was observed. The whole procedure takes less than 6 h and can also be used as a semipreparative method for high-affinity heparin.     

Quantitative measurement of lipoprotein surface charge by agarose gel electrophoresis.

The electrophoretic mobilities of low density lipoprotein (LDL) and six pure proteins in a 0.5% agarose gel have been compared to literature electrophoretic mobility values determined by the Tiselius moving boundary method. There is a strong correlation (r = 0.99) between the electrophoretic mobilities determined by the two techniques. The electrophoretic behavior of charged particles smaller than very low density lipoproteins (VLDL) is not markedly perturbed by a 0.5% agarose matrix, and variations in mobility primarily reflect differences in particle valence and density of surface charge. Application of electrokinetic theory to derive protein and lipoprotein net charges from the electrophoretic mobilities in agarose yields a quantitative delineation of lipoprotein electrophoretic migration patterns wherein the beta mobility region comprises a surface potential range of -4.5 to -7.0 mV; the pre-beta region a range of -7.0 to -10.5 mV; the alpha mobility region a range of -10.5 to -12.5 mV and the serum albumin region a range of -12.5 to -14.0 mV. Because protein conformation and charge are critical in metabolic regulation, the agarose gel electrophoresis technique provides a valuable analytical tool that should help to elucidate further details of the structure-function relationships of serum lipoprotein particles.     

Cell microelectrophoresis simplified by the reduction and uniformization of the electroosmotic backflow

By coating the inside of the electrophoresis capillary with an agarose gel and by plugging its ends with the same material, the electroosmotic backflow is much reduced and simultaneously acquires a uniform velocity at all levels within its lumen. After taking into account a small electroosmotic correction factor, the true electrophoretic mobility of cells in an electric field can directly be determined, at any level of focusing. This greatly simplifies the microelectrophoretic method.     

Preparative agarose gel electrophoresis for the purification of small organelles and particles

A novel preparative method of quantitative flatbed agarose gel electrophoresis has been used to separate a number of small subcellular structures, such as ribosomes, coated vesicles, smooth vesicles, and ferritin. The technique utilizes continuous elution of a second, electrophoretically "downstream," well in the agarose gel. The elution occurs concurrently with the electrophoresis, so essentially no additional time is required for the recovery of the structures. The technique is nondestructive, relatively simple and inexpensive, and can be used by modifying any nonsubmerged horizontal agarose gel system. The preparative separation of small organelles and subcellular structures according to their charge allows the purification of small structures previously difficult to isolate by conventional techniques. Two novel structures purified by this technique are described: a short intermediate filament-like species consisting of a single polypeptide of Mr 142,000, and an ovoid species (70 X 35 nm) whose protein composition is dominated by a polypeptide of Mr 104,000.     

Purification and characterization of bovine lipoproteins: resolution of high density and low density lipoproteins using heparin-Sepharose chromatography

The selective and reversible adsorption of bovine low density lipoproteins (LDL) by heparin-Sepharose has been exploited as the critical step in a procedure for the preparative isolation of very low density lipoproteins (VLDL)/chylomicrons, LDL, and high density lipoproteins (HDL) from bovine plasma. Molecular size exclusion chromatography and isopycnic density gradient separation steps are also involved in the method described. The resulting HDL and LDL fractions are free from contamination by one another as judged by electrophoretic mobility in agarose gels. The major lipid and apolipoprotein compositions of the three resolved lipoprotein classes have been determined.     

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple immunoreplica Technique: screening for specific proteins with a series of different antibodies using one polyacrylamide gel

A simple method for immunological identification of proteins resolved electrophoretically is presented. Proteins from one polyacrylamide gel can be subjected to a series of electrophoretic transfers to nitrocellulose paper (partial “western-blots”), providing several replicas of the gel. Each replica can be reacted with a series of different antisera (at least three), where the preceding antibody is removed by treatment with pH 2.2. The antigen-antibody complexes are visualized using ¹²⁵I-Protein A. Reactivity and antigenic specificity of proteins immobilized on nitrocellulose paper is not affected by repeated incubations and low pH treatments. Identical size of the replicas and superimposable profiles of proteins detected by antibodies allow a precise localization of particular polypeptides in the original gel.     

Labelling of colloidal gold with protein A. A quantitative study

Colloidal gold complexes with protein A are extensively used in immunocytochemistry as secondary reagents for the localization of antigens. However detailed information on the process and extent of adsorption of protein A onto gold particles, the optimal condition of preparation and the stability of such complexes are lacking. The adsorption isotherm of 125I-protein A onto gold particles (11.2 nm in diameter) was studied quantitatively with gold sols buffered at pH 4-7. At low coverage of the particles, the isotherm was independent of pH. However in the presence of a large excess of protein A, the highest coverage was obtained with a gold sol buffered at pH 5.1, the isoelectric point of the protein. The association constant was decreased at high coverage of the particles. Maximum binding of the complex to immobilized IgG occurred with particles labelled with at least 9 molecules of protein A. The complex was stable under storage with up to 12 molecules adsorbed per particle. At high coverage (26 molecules per particle), a progressive loss of protein A was observed. The optimum condition for preparing the complex are reported.     

Determination of a particle's radius by two-dimensional agarose gel electrophoresis

Electrophoresis in an agarose gel dilute enough to be almost nonretarding, followed by electrophoresis in an orthogonal direction into a more concentrated agarose gel, has been developed as a procedure to determine the radius of spherical particles. Unlike procedures of unidirectional electrophoresis in a single gel, the above procedure can be used to compare the radii of particles that differ in solid-support-free electrophoretic mobility. Accuracy of 0.3 nm has been achieved with particles 30 nm in radius. It was found that the apparent radius of the spherical capsid of bacteriophage P22 decreased by 3% during elevated temperature-induced ejection of DNA from the capsid. Though originally designed for use with multimolecular particles, the procedure described here should also be useful with monomolecular particles.     

Studies on the altered electrophoretic type of the factor VIII related antigen

Summary A distinct sub-group of von Willebrand's disease is characterized by an electrophoretically faster mobility of the factor VIII related antigen. Some of the physico-chemical properties of this variant antigen were investigated in this communication. The effect of temperature was tested by heating aliquots (0.5 ml) for 20 minutes at 45°C, 56°C and 65°C. The variant was found to be denatured at 56°C while the control was denatured at 65°C. The effect of pH was tested by assessing the quantity (Laurell technique) and electrophoretic mobility (two dimensional immunoelectrophoresis) of the antigen in a variety of buffers ranging in pH from 7.0 to 9.5. The quantity of antigen was variable both among variants and controls and the electrophoretic mobility of the variant antigen was faster at all pH's. Molecular weight differences between the variant and controls were not detected since the chromatographic profile of the variant was similar to that of the controls in Sepharose 6 B using a 0.02 M Tris-NaCl buffer at pH 7.0. The affinity of the antigen for human antibody was geterogeneous although the variant exhibited less affinity for one of the human antibodies but not the other. The inhibitory effect was more pronounced in serum than in plasma. Purified IgG, however, did not show any inhibition, as the residual antigen assayed by the Laurell technique, was similar to the expected values. This would imply that non-IgG plasmatic factors could also play a part in the observed inhibition.     

Fluorographic detection of [3H]thymidine-labeled deoxyribonucleic acid using agarose gels infused with 2,5-diphenyloxazole prior to electrophoresis

A fluorographic procedure for the detection of [3H]thymidine-labeled deoxyribonucleic acids electrophoresed in agarose gels was developed. 2,5-Diphenyloxazole (PPO) was added to the agarose solution before pouring of the gel for electrophoresis. This procedure did not interfere with the electrophoretic mobility of the DNA molecules. The radioactive detection efficiency was found to be improved over an existing procedure whereby the agarose gel was infused with PPO after electrophoresis with the aid of acetic acid.     

Effective diameters of protein A-gold and goat anti-rabbit-gold conjugates visualized by field emission scanning electron microscopy.

High-voltage (15-30 kV) field emission scanning electron microscopy (FESEM) was used to evaluate the effects of gold particle size and protein concentration on the formation of protein-gold complexes. Six colloidal gold sols were prepared, ranging in diameter from 7.6 to 39.8 nm. The minimal protecting amounts (m.p.a.) of protein A and goat anti-rabbit antibody (GAR) were experimentally determined. Gold particles were conjugated at the m.p.a., one half the m.p.a., and ten times the m.p.a. for both proteins, and protein-gold complexes prepared for FESEM. The smallest colloidal gold particles required the most protein per milliliter of gold suspension for stabilization. Transmission electron microscopy was found to be the preferred method for accurate sizing of gold particles, whereas FESEM of protein-gold complexes permitted visualization of a protein halo around a spherical gold core. Protein halo width varied significantly with changes in gold particle size. Measurements of protein halos indicated that conjugation with the m.p.a. of protein A resulted in the thickest protein layers for all gold sizes. GAR conjugation with the m.p.a. again produced the thickest protein layers. However, GAR halos were significantly smaller than those obtained with protein A conjugation. The proteins used showed similar adsorption patterns for the larger gold particles. For smaller gold particles, proteins may act differently, and these complexes should be further characterized by low-voltage FESEM.     

Creatine kinase isoenzyme associated with synaptosomal membrane and synaptic vesicles

Brain creatine kinase is principally of soluble cytoplasmic origin (anodal electrophoretic mobility). However, synaptosomal membranes and synaptic vesicles are enriched in an isoenzyme electrophoretically similar to muscle type creatine kinase (cathodal electrophoretic mobility), but which can be distinguished from muscle type by other means.     

A method for horizontal polyacrylamide slab gel electrophoresis

We present a simplified method of preparation of polyacrylamide gels which is totally analogous to the procedure now widely used to pour and run horizontal agarose gels. The acrylamide is poured into an open air gel mold consisting of a glass plate with a masking tape border and a comb. It is subsequently run in a submarine horizontal electrophoresis apparatus. The electrophoretic mobility and resolution of DNA fragments obtained in such gels are identical to results obtained with gels poured and run in the vertical configuration. Numerous advantages of horizontal polyacrylamide gel electrophoresis are discussed.     

Zonal immobilization of proteins

A gel matrix that can be used in sequence to separate proteins and then immobilize them was obtained by incorporating into agarose an aldehydic ligand with readily controllable reactivity. The gel was prepared by etherifying agarose with glycidol and subsequently oxidizing with periodate. It provided an inert matrix equivalent to ordinary agarose for separating proteins at neutral or acidic pH, but rapidly absorbed them through formation of stable alkyl amine linkages on exposure to either alkaline or concentrated NaCNBH₃. Thus, the protein could be fixed without use of denaturants. The ability to array proteins electrophoretically on an immobilizing substrate opens new possibilities for analysis of complex mixtures by providing means for carrying out affinity binding assays in relation to physical properties of the protein, and for performing multiple tests of composition without loss or spread.     

Gradient acrylamide/agarose gels for electrophoretic separation of intact human very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins

An exponential gradient gel with 0-10% acrylamide and 0.5% agarose was developed for electrophoresis of intact high molecular weight lipoproteins. This system resolves very low density lipoproteins, intermediate density lipoproteins, lipoprotein a, and low density lipoproteins in a size-dependent fashion. The characteristic relative mobility of these species can be determined in relation to protein and colloidal gold reference materials. Electron microscopy of selected lipoprotein fractions confirmed that relative mobility was related to apparent lipoprotein diameter. The composite gel medium can be used with prestained lipoproteins and permits immunoelectroblotting for qualitative analysis of apolipoprotein constituents.