Gel Electrophoretic Method Research Articles

Multiple proteases from Streptomyces moderatus: I. Isolation and purification of five extracellular proteases

The presence of multiple proteases in the culture filtrate of Streptomyces moderatus was detected. After preliminary purification by ammonium sulfate precipitation and decolorization using DEAE-cellulose, the fractionation of various proteases was carried out using CM-trisacryl cation-exchange chromatography. By this procedure, four different protease fractions (Fr.) were separated (Fr. I, II, III, and IV). The first fraction was further separated into two different proteolytically active fractions (Fr. Ia and Fr. Ib) by DEAE-trisacryl anion-exchange chromatography. Fraction Ia was purified further by affinity chromatography on N-carbobenzoxy- d-phenylalanyl triethylenetetramine-Sepharose 4B. The second fraction (Fr. Ib) was purified by gel filtration on Ultrogel AcA 44. For the purification of the other protease fractions (Fr. II, III, and IV) single-step affinity chromatography methods were employed. Protease fractions II and III were purified by ϵ-aminocaproyl-4-(4-aminophenylazo)phenylarsonic acid Sepharose 4B and protease fraction IV was purified on ϵ-aminocaproyl trialanine-Sepharose 4B. All five proteases purified were found to be apparently homogeneous by gel electrophoretic methods.

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.

A regulatory polymorphism for rat hepatic cytochrome P-450g

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F 1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cisacting.

Ｔｉｓｓｕｅ‐ｐｌａｓｍｉｎｏｇｅｎ Ａｃｔｉｖａｔｏｒによる血栓治療 Ｇｌｙｏｘｙｌ ａｇａｒｏｓｅ ｇｅｌ電気泳動による検討

We studied on detection of plasma fibrinogen derivatives of tissue-Plasminogen Activator (t-PA) mediated thrombosis with Glyoxyl agarose gel electrophoresis. Plasma proteins were immobilized into aldehyde-rich Glyoxyl phoresis. Plasma proteins were immobilized into aldehyde-rich Glyoxyl agarose with NaCNBH3 after electrophoresis, and immobilized fibrinogen derivatives were blotted with fluorescent anti-fibrinogen. Fibrinogen derivatives [Fibrin monomer, polymer (HMW); Fibrinogen (φ); Fragment X, Y (φ′); Fragment D, E (LMW)] were detected by the mobility on the gel and their concentrations were calculated by the fluorescent intensity of them. Standard plasma had no fluorescent blotting peak by this method. The change of fibrinogen levels of t-PA-mediated thrombosis (total 13 patients) were classified into 3 groups; Slightly lysis (7/13), Mild lysis (3/13), Systemic lysis (3/13) and the electrophorogram of thrombosic patient plasma was resemble to that of malignant hypertention rat, but only a few normal human had the HMW peak (1/7). It was concluded that Glyoxyl agarose gel electrophoretic method was very useful and sensitive for the detection of fibrinogen derivatives with small sample. This method could be used for the clinical samples, because immobilized proteins into gel were stable and possible to store for a few weeks.

Characterization of proteins from mitochondrial ribosomes of Locusta migratoria by three different two-dimensional gel electrophoretic methods and comparison with cytoplasmic ribosomal proteins

1. 1. Proteins were isolated from subunits of mitochondrial and cytoplasmic ribosomes of Locusta migratoria and were analyzed by means of two-dimensional gel electrophoreses using three different electrophoresis systems. 2. 2. Using the system of Czempiel et al. (1976) proteins from whole locust mitochondrial ribosomes (combined subunits) were separated into 72 spots; proteins from the large and small subunits resulted in 48 and 29 spots respectively. 3. 3. The mol. wt distribution of mitochondrial ribosome proteins was estimated by using the electrophoresis system of O'Farrell (1975). These mol. wts are in the range of 11,000–56,000, the average mol. wt is about 29,500. Assuming one copy of protein per ribosome this gives a total mol. wt for the protein part of mitochondrial ribosomes of ca. 2.1 x 10 6. 4. 4. Parallel separation of cytoplasmic and mitochondrial ribosome proteins was achieved using the system of Geyl et al. (1981). Cytoplasmic ribosome proteins produced 65 spots and revealed a more alkaline character than mitochondrial ribosome proteins.

Improved banding pattern of rat plasma lipoproteins developed by agarose gel electrophoresis at pH 7.0

The use of a conventional agarose gel electrophoretic method to separate rat plasma lipoproteins resulted in a rather poor resolution of lipoproteins of lower density. Therefore, attempts were made to improve the resolution by running the electrophoresis under different conditions. It was shown that rat plasma lipoproteins could be separated into at least three fractions by agarose gel electrophoresis in phosphate buffer at pH 7.0. Using rat plasma lipoproteins isolated by sequential flotation as standards, these fractions were shown to correspond to high-density lipoprotein (HDL 2), very-low-density lipoprotein (VLDL) and a mixed low-density lipoprotein (LDL)/high-density lipoprotein (HDL 1) fraction. Since VLDL is completely separated from the other lipoprotein classes the method could be used to monitor changes in plasma VLDL.

Characterization of the filamentous hemagglutinin from Bordetella pertussis by gel electrophoresis.

A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S1 and S2 (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form Ib, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S2, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.

KDNA minicircles of the major sequence class of C. fasciculata contain a single region of bent helix widely separated from the two origins of replication.

The major sequence class of Crithidia fasciculata minicircles is shown to have a single region of bent helical DNA widely separated from the two replication origins located 180 degrees apart on the minicircle map. The position of the bend in the DNA has been mapped both by gel electrophoretic methods and by direct electron microscopic observation of the DNA. This sequence directed bending is apparently the result of homopolymeric dA X dT tracts 4-6 base pairs long repeated in phase with the helix screw. The region of the bend contains nineteen such homopolymeric tracts in a region of about 200 base pairs with sixteen of the tracts oriented in the same direction.

Quantitation of Apo-, Mono-, and Diferric Transferrin by Polyacrylamide Gradient Gel Electrophoresis in Patients With Disorders of Iron Metabolism

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.

Quantitation of apo-, mono-, and diferric transferrin by polyacrylamide gradient gel electrophoresis in patients with disorders of iron metabolism

We have developed a polyacrylamide gradient gel electrophoretic method to quantitate apo-, mono-, and diferric transferrin based upon differences in their molecular size. Purified transferrin saturated to different extents (3% to 98%) with iron showed proportions of the three forms as predicted from an approximately random distribution of iron between the two metal-binding sites. The iron distributions in sera of 14 normal individuals similarly correlated with the predicted values. In contrast, 22 of 43 patients with diseases associated with abnormalities in iron or transferrin metabolism had a disproportionate increase in monoferric transferrin. This abnormality occurred in seven of nine patients who had received bone marrow transplants, seven of 14 with chronic liver disease, and eight of nine menstruating women with probable iron deficiency anemia. Interestingly, 11 patients with malabsorption or chronic renal disease had normal iron distributions. The finding of abnormal distributions of iron on transferrin suggests that gradient gel analysis may be a useful tool for studying the physiologic mechanisms controlling iron utilization.

An Agarose Gel Electrophoretic Method for Typing Phosphoglucomutase-1, Esterase D, or Glyoxalase I

A conventional agarose gel electrophoretic method was described for typing phosphoglucomutase-1, esterase D, or glyoxalase I as single systems. Bloodstain extracts were absorbed into 1-mm-thick agarose gels via an application mask. The electrode wick distance was 12 cm and electrophoresis was carried out at 400 V at 6 degrees C. The electrophoretic run times were 30 min for glyoxalase and 1 h for esterase D or phosphoglucomutase. This method is reliable and produces highly resolved band patterns. Additionally, the shorter separation times as a result of the increased voltage gradient permitted typing of more samples in a given time period compared with presently used methods. This technique requires little technical expertise and can be incorporated into the laboratory at a minimal cost.

Heat‐Induced Aggregation and Denaturation of Egg White Proteins in Acid Media

ABSTRACTHeat‐induced aggregation and denaturation of egg white proteins adjusted to pH 5.5, 4.5, 3.5 and 2.5 were investigated by vertical flat‐sheet polyacrylamide gel electrophoretic and differential scanning calorimetric methods. The fractional and step‐wise aggregation of egg white proteins was caused by heating. As the acidity was increased from pH 5.5 to 2.5, ovotransferrin, ovomacroglobulin, globulin G3A, globulins A1 and A2, and ovalbumin became much more unstable to heat. However, ovomucoid and ovoinhibitor did not aggregate in the acidified egg white under heat treatments of 3 min at 90°C or 20 min at 74°C. The heat‐induced aggregation of flavoprotein was slightly greater at pH 4.5 and 3.5 than at pH 5.5 and 2.5.

Rapid Separation and Quantification of Major Caseins and Whey Proteins of Bovine Milk by Polyacrylamide Gel Electrophoresis

A rapid polyacrylamide gel electrophoretic method was developed for separating and quantifying major proteins in casein and whey protein fractions of bovine milk. For casein separation, best results were achieved by an 8% polyacrylamide gel containing 4 M urea and a top layer of large pore sample gel; for whey protein the most satisfactory separation was with 12% polyacrylamide gel in the absence of urea and a large pore gel. Electrophoretic conditions for separation of whey protein were also applicable for identifying genetic variants of β-lactoglobulin. Densitometric tracings were used to resolve and quantify protein peaks from stained bands in the gels. Casein was resolved into three major fractions with relative proportions of 50:30:15 for αs1- and αs2-casein, β-casein, and κ-casein. Whey protein was resolved into four major fractions with relative proportions of approximately 60:20:7:13 for β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulin.

Increase in fast hemoglobin concentration after incubation with acetaldehyde detected by an electrophoretic method.

Incubation of acetaldehyde with hemoglobin has been shown by chromatographic methods to result in an increase in fast hemoglobin. We have studied this increase in fast hemoglobin concentration by a gel electrophoretic method. Increases in fast hemoglobin concentration are detected following incubation with concentrations of 100 microM acetaldehyde and no further increase in yield occurs with concentrations greater than 7 mM acetaldehyde. The reaction is complete within 5 hr and the product is stable with a half-life of greater than 12 days.

A gel electrophoresis method for epitope mapping studies with monoclonal antibodies

The linear polyacrylamide gradient gel electrophoresis method of Lambin and Fine (1979, Anal. Biochem. 98, 160–168) has been adapted for estimation of the molecular weights of antigen-antibody complexes, thereby providing information useful in epitope maping studies with monoclonal antibodies. The method has been applied to mapping of the epitopes recognized by four different monoclonal antibodies raised against rat brain hexokinase (1984, Finney et al., J. Biol. Chem., 259, 8232–8237). The results obtained with the gel electrophoresis method were in agreement with epitope mapping studies conducted by the molecular sieve high-performance liquid chromatographic method of Crawford et al., (1982, Proc. Natl. Acad. Sci. USA 79, 7031–7035). Epitope mapping by the gel electrophoretic method offers several advantages in terms of speed, convenience, and economy when compared with alternative procedures that have been described.

Purification of the photoaffinity-labeled glucagon receptor by gel electrophoretic methods.

Rat liver plasma membrane glucagon receptor has been purified with a yield of 0.01% to an estimated homogeneity of 32-60%, using a 2-stage electrophoretic procedure. SDS-solubilized membrane proteins labeled by the photoaffinity-agent, Ne-4-azidophenylamidinoglucagon (APA-glucagon), were separated by polyacrylamide gel electrophoresis in SDS-containing buffers. Gel slices corresponding to the molecular weight of the receptor were excised, electrophoretically extracted and concentrated. The concentrate was subjected to isoelectric focusing on Sephadex to yield a purified product in which the photoaffinity-labeled receptor, with a molecular weight of 56K and a pI' of 5.9, is the sole major component.

Isoelectric focusing followed by silver staining. A suitable method for routine investigation of cerebrospinal fluid proteins.

Cerebrospinal fluid (CSF) and serum samples from 361 consecutive patients were investigated. The protein pattern of each sample was examined by isoelectric focusing (IEF) of unconcentrated CSF followed by silver staining and by agar gel electrophoresis of concentrated CSF stained with amido black. 347 paired samples were compared. The results were the same in 83% of the samples. Discordant results were found in 10% of the samples. and in 7% additional information was achieved by one of the methods. IEF was superior to agar gel electrophoresis for the identification of oligoclonal immune reactions of the central nervous system. Tau globulin increase was seen in a few CSF samples, more often with IEF and then associated with other changes of the pattern. IEF of unconcentrated CSF followed by silver staining only requires 20 microliters CSF, while 3-4 ml CSF is needed for the agar gel electrophoretic method. It was concluded that IEF has several advantages compared to agar gel electrophoresis and is suitable as a routine method in clinical laboratories.

IDENTIFICATION OF CICER MILKVETCH CULTIVARS USING SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS

With the release of Monarch cicer milkvetch (Astragalus cicer L.), which has greatly facilitated stand establishment when compared to older cultivars, the desirability of a method for identifying seed of cultivars was recognized. An SDS-polyacrylamide gel electrophoretic method was developed to differentiate cicer milkvetch cultivars. Densitometric scanning of the resultant polypeptide banding pattern on the polyacrylamide gel provided identification of seed lots of the cultivars Oxley, Lutana and Monarch produced in several different years and locations.Key words: Astragalus cicer L., forage legumes, seed proteins, varietal purity

Quantitative analysis of the association of human hemoglobin with the cytoplasmic fragment of band 3 protein.

The association of the isolated cytoplasmic fragment of band 3 protein with human hemoglobin was studied by rate zonal centrifugation in sucrose density gradients, by quenching of fragment fluorescence by hemoglobin, and by flash photolysis of carbon monoxidebound hemoglobin as a function of fragment concentration. The centrifugation results showed that both proteins interact and that the interaction is abolished upon addition of glyceraldehyde-3-phosphate dehydrogenase. The fractions eluted from the density gradient were analyzed further by spectrophotometric and gel electrophoretic methods. Two types of complexes could be identified, one containing the equivalent of 1 hemoglobin tetramer/dimer of cytoplasmic fragment and another containing 2 tetramers of hemoglobin/dimer of fragment. Flash photolysis and fluorescence-quenching experiments showed that liganded hemoglobin is stabilized as the alpha beta dimer when bound to the fragment, a result almost identical with that seen for membranebound hemoglobin in previous studies. The results further suggest that there are two binding sites for the alpha beta dimer of hemoglobin on one monomer of the cytoplasmic fragment but only one mutually exclusive hemoglobin tetramer binding site, suggesting the possibility of conformational isomerism when the fragment with two dimers bound isomerises to a fragment monomer with one hemoglobin tetramer bound. Finally, despite the stabilization of the dimeric state of liganded hemoglobin when bound to the fragment, estimates of the hemoglobin dimer and tetramer binding constants suggest that the hemoglobin tetramer binds more tightly by about 2 orders of magnitude.

Determination of Added Water and Bovine Milk to Caprine Milk

Increased production of commercial goat milk in Ontario stimulated an investigation to determine a base freezing point for goat milk for quantifying added water. A method also was developed for detection and estimation of degree of adulteration of goat milk with bovine milk. The mean freezing point based on 228 fresh herd samples of goat milk taken over 1 yr was −.5527°C (−.5719°H). This mean was used to generate an added water table. A simple and direct polyacrylamide disc gel electrophoretic method was devised to determine semi-quantitatively bovine milk adulteration down to 5%. Both of these methods are currently in use for routine monitoring of goat milk adulteration in the province of Ontario.