Mixed-dye staining method for protein detection in polyacrylamide gel electrophoresis using calconcarboxylic acid and rhodamine B

We have developed a new mixed-dye protein staining method that is simple, rapid, and sensitive. A freshly prepared mixture of calconcarboxylic acid (NN, 0.02%) and rhodamine B (RB, 0.04%) in 40% methanol/7% acetic acid, was used as a staining solution. RB acts as an auxiliary agent to inhibit the binding of NN to the gel matrix, reducing the background staining and therefore enhancing the protein staining by NN. This mixed-dye staining method reduces the total staining and destaining time to less than an hour, and increases the sensitivity to 25 ng of bovine serum albumin, which is greater than the 100 ng sensitivity limit of Coomassie Brilliant Blue R-250 (CBBR) staining.     

Correlation of plasma protein separation patterns obtained by two-dimensional polyacrylamide gel electrophoresis and by capillary electrophoresis

We used three different electrophoretic techniques for the analysis of human plasma proteins: (i) two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), with sodium dodecyl sulfate (SDS) used only in slab gel electrophoresis; (ii) capillary isoelectric focusing (CIEF) with no denaturants; (iii) linear polyacrylamide (LPA)-filled capillary electrophoresis with SDS (SDS-CE). With technique (i), data on isoelectric point and molecular size of plasma proteins can be obtained. Techniques (ii) and (iii) are suited to obtain quantitative information on proteins. The separation principle used in technique (ii) is closely related to that used in the first dimension of technique (i), and that used in technique (iii) related to that in the second dimension of technique (i). Therefore, we could successfully correlate protein separation patterns obtained by 2-D PAGE and those obtained by capillary electrophoresis. The advantages of correlating data obtained by various electrophoretic techniques in the course of constructing a comprehensive database on human plasma proteins are discussed.     

Polypeptidic composition analysis of "Chlamydia" purified elementary bodies by polyacrylamide slab gel electrophoresis (author's transl)

The study of the polypeptidic components of elementary bodies of various strains of Chlamydia might help in the classification of these bacteria. The use of a satisfactory method for the preparation of purified elementary bodies has permitted the electrophoretic analysis of their crude lysate on polyacrylamide gel in the presence of sodium dodecyl sulfate. Differences between the strain LB 1 C. trachomatis and the strains Loth and MP 1 C. psittaci were found.     

Rapid characterization of mutations in amplified human hprt cDNA by polyacrylamide gel electrophoresis

Local cerebral serotonin synthesis capacity was measured with alpha-[C-11]methyl-L-tryptophan ([C-11]AMT) in normal adult human brain (n = 10; five males, five females; age range, 18-38 years, mean 28.3 years) by using positron emission tomography (PET). [C-11]AMT is an analog of tryptophan, the precursor for serotonin synthesis, and is converted to alpha-[C-11]methyl-serotonin ([C-11]AM-5HT), which is trapped in serotonergic neurons because [C-11]AM-5HT is not degraded by monoamine oxidase. Kinetic analysis of [C-11] activity in brain after injection of [C-11]AMT confirmed the presence of a compartment with unidirectional uptake that represented approximately 40% of the activity in the brain at 50 min after tracer administration. The undirectional rate constant K, which represents the uptake of [C-11]AMT from the plasma to brain tissue followed by the synthesis and physiologic trapping of [C-11]AM-5HT, was calculated using the Patlak graphic approach on a pixel-by-pixel basis, thus creating parametric images. The rank order of K values for different brain regions corresponded well to the regional concentrations of serotonin in human brain (P < .0001). High serotonin synthesis capacity values were measured in putamen, caudate, thalamus, and hippocampus. Among cortical regions, the highest values were measured in the rectal gyrus of the inferior frontal lobe, followed by transverse temporal gyrus; anterior and posterior cingulate gyrus; middle, superior, and inferior temporal gyri; parietal cortex; occipital cortex, in descending order. Values in women were 10-20% higher (P < .05, MANOVA) throughout the brain than those measured in men. Differences in the serotonin synthesis capacity between men and women measured in this study may reflect gender differences of importance to both normal and pathologic behavior. This study demonstrates the suitability of [C-11]AMT as a tracer for PET scanning of serotonin synthesis capacity in human brain and provides normal adult values for future comparison with patient groups.     

Development of a quantitative polyacrylamide gel electrophoresis analysis using a multichannel radioactivity counter for the evaluation of oligonucleotide-bound drug carrier

A quantitative polyacrylamide gel electrophoresis (PAGE) analysis using a multichannel radioactivity counter was designed for the evaluation of 33P-labeled antisense oligonucleotide associated with polymeric drug carrier (nanoparticles). The proposed analytical method was first validated. The criteria of specificity, linearity, reliability, detection and quantification limits, and resolution power were determined. Results were compared to those obtained using liquid scintillation counting of crude samples or after solubilization of gel slices. The proposed method gave a better linearity and reliability than liquid scintillation counting of solubilized gel slices. In comparison with the liquid scintillation counting of crude samples, the method presented the advantage of being able to directly separate oligonucleotides differing by only one nucleotide in length. This method was applied for the separation of free oligonucleotides and oligonucleotides bound onto nanoparticles, allowing quantification of the amount of free and bound oligonucleotides without any further separation steps. Thus, because it is easy and rapid, the quantitative PAGE analysis using a multichannel radioactivity counter offers interesting possibilities for the characterization of oligonucleotide nanoparticles.     

Isolation and characterization of beta-cyclodextrin sulfates by preparative gradient polyacrylamide gel electrophoresis, capillary electrophoresis and electrospray ionization - mass spectrometry

A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.     

Importance of sodium dodecyl sulfate pore-graduated polyacrylamide gel electrophoresis in the differential diagnostic of Balkan nephropathy

The reliance on earthworms as test organisms in risk assessment studies of polluted environments raises the question whether they can evolve resistance, e.g., by adaptation to specific toxicants. Protection criteria may be biased if sensitivity data from adapted populations are used. Increased resistance to the heavy metal cadmium has not yet been determined for terrestrial Oligochaeta. Eisenia fetida was exposed to a sublethal concentration of cadmium sulfate for more than 10 generations. Clitellate worms from this culture were used in experiments to determine the extent of possible tolerance for the heavy metal. Preexposed animals as well as worms with no previous history of exposure to cadmium were exposed to a control substrate without cadmium and also to two substrates with 600 and 1200 microg g-1 cadmium. Changes in biomass, cocoon production, and hatching success were monitored. The results obtained indicated that in both substrates in which cadmium was present the preexposed worms performed better than the unexposed worms with respect to growth rate but not reproductively. In the substrate without cadmium the preexposed worms exhibited signs of poisoning after a few weeks. Preexposed and unexposed worms were also exposed to concentrations of 1500 to 4000 microg g-1 cadmium sulfate in an artisol medium for a period of 2 weeks. The preexposed worms survived higher concentrations of cadmium than the unexposed group and some specimens from the unexposed group had a gross increase in body fluids. It is concluded that worms with a long-term history of exposure to the metal developed resistance to cadmium.     

The resolution between two native proteins and between their sodium dodecyl sulfate-complexes in agarose and polyacrylamide gel electrophoresis

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE-1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG-, SeaKem Gold-agarose, 2% N,N'-methylenebisacrylamide cross-linked polyacrylamide, MetaPhor-XR-, and SeaPrep-agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4-6% SeaPrep agarose than in 4-6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS-proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards - proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3-8%, and that therefore the molecular "fit" in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius ("fit") is also suggested by the fact that the two SDS-proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.     

Partition and permeation of dextran in polyacrylamide gel

Partition of sized FITC-dextrans in polyacrylamide gel showed a relationship between Kav and solute radius as predicted by the theory of Ogston, which is based solely on geometry of the spaces. Permeability data for the same dextrans were fit to several theories, including those based on geometry and those based on hydrodynamic interactions, and the gel structure predicted by the partition and permeability data were compared. The Brinkman effective-medium model (based on hydrodynamic interactions and requiring a measure of the hydraulic conductivity of the matrix) gave the best fit of permeability data with the values for fiber radius (rf) and void volume of the gel (epsilon) that were obtained from the partition data. The models based on geometry and the hydrodynamic screening model of Cukier, using the rf and epsilon from partition data, all predicted higher rates of permeation than observed experimentally, while the effective-medium model with added term for steric interaction predicted lower permeation than that observed. The size of cylindrical pores appropriate for the partition data predicted higher rates of permeation than observed. These relative results were unaffected by the method of estimating void volume of the gel. In sum, it appears that one can use data on partition of solute, combined with measurement of hydraulic conductivity, to predict solute permeation in polyacrylamide gel.     

Recovery of SDS-protein and DNA using commercial automated gel electrophoresis apparatus

Forty-seven children afflicted with acute leukemia were studied at the Tata Memorial Hospital Bombay to record the occurrence of oral manifestations prior to and during chemotherapy. Lymphadenopathy was the most frequent single finding suggestive of leukemia during head and neck examination. Gingival abnormalities, bleeding gums and oral mucosal pallor were the other findings on initial oral examination. Due to immunosuppression caused by the chemotherapy drugs oral mucosal ulcerations, uncontrolled herpes, candidiasis and pseudomoniasis were observed.     

Analysis of the low molecular weight nuclear polypeptide by isoelectrofocusing of nuclear proteins first separated by gel electrophoresis in SDS

Some of the nuclear proteins, especially those tightly bound with nucleic acids, show a tendency to aggregate and some of them are also hydrophobic. Thus, SDS-containing buffers are useful for their analysis. The method of isoelectrofocusing of proteins separated by polyacrylamide gel electrophoresis in the presence of SDS was used for the analysis of the nuclear proteins. First, SDS-polyacrylamide gel electrophoresis of total chromatin proteins was conducted. The sample of the examined nuclear proteins after SDS-gel electrophoresis was isolated as a gel slice (thus corresponding to the particular molecular weight range) and used for isoelectrofocusing. This approach has been used for the analysis of about 19 kD nuclear polypeptide, tightly bound with DNA. The isoelectric point of this polypeptide has been estimated as 5.3.     

Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.     

Classification of Setaria worms from calves and adult cattle into Setaria digitata and Setaria marshalli by polyacrylamide gel electrophoresis

The protein profiles of solubilized whole worms of the species, Setaria digitata and Setaria marshalli were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results showed that one band with a molecular size of 69 kDa was confirmed only in S. marshalli, while this band was not detected in S. digitata. There were no differences of the major bands between males and females of the respective worm species. As further investigation, two-dimensional gel electrophoresis was performed and at least one spot ranging from 64 to 73 kDa was confirmed in S. marshalli but not in S. digitata. Therefore, those worms could be classified into two species biochemically as well as by the morphological characteristics.     

The influence of sodium dodecyl sulfate from different sources on the separation of virus proteins in polyacrylamide gel electrophoresis

Bacteriophage 7-7-1 is shown to adsorb specifically to the complex flagella of its host Rhizobium lupini H13-3. Deflagellation of motile cells before the addition of phage leads to a complete inhibition of phage propagation for at least 60 min. Among phage-resistant mutants, many non-motile (mot) and non-flagellated (fla) derivatives of R. lupini H13-3 have been selected. Electron microscopic observations indicate that bacteriophage 7-7-1 attaches with its short tail fibres to the conspicuous helical filament of R. lupini flagells. This attachment is reversible; irreversible phage adsorption takes place at the flagellar base. It is postulated that phage 7-7-1 moves along the rotating flagellum towards a final receptor next to the insertion site of the flagellum, where tail contraction and injection of phage nucleic acid occurs.     

An accurate determination of human growth hormone content in different pituitary extracts, using a radioimmunoassay with polyacrylamide gel electrophoresis as a bound-free separation system

Human growth hormone was extrated and purified according to the method of Roos et al. (Roos, P., Fevold, H.R. and Gemzell, C.A. (1963) Biochim. Biophys. Acta 74, 525). A first control of its purification and integrity was performed through molecular weight determination by gel filtration on Sephadex G-100 and on polyacrylamide gel electrophoresis (PAGE). Its biological activity was confirmed by the growth promoted in non-hypophysectomized rats at plateau. The main object, however, was the setting up of accurate, reproducible method tha could furnish the more absolute and comparable values of radioimmunoassayable HGH content in perfect agreement with the results obtained by other laboratories. This was accomplished through a radioimmunoassay system that uses HGH labelled with 125I, where separation of the bound from the free antigen is achieved on polyacrylamide gel electrophoresis, by a modification introduced in the original method of Davis. The resulting values, extremely close to that stated by the KABI-Laboratories (Stockolm), though obtained in quite different conditions of incubation, antibody concentration and with no use of second antibody, represent a confident approach to a comparable measure of this hormone in extracts, which can also be applied to plasma determinations.