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.     

Improved sensitivity of detection with the commercial automated gel electrophoresis (HPGE-1000) apparatus through modification of its optical system

In a representative application to a fluorescently detectable protein of commercial automated gel electrophoresis apparatus (HPGE-1000, LabIntelligence, Belmont, CA) the sensitivity of detection by fluorescence was significantly increased by elimination of the mirror below the gel tray. That increase in detection sensitivity is due to a decrease of fluorescent background noise by nearly one order of magnitude, overcompensating a decrease in signal by a factor of two. The resulting increase in signal/noise ratio, i.e., detection sensitivity, should allow for lowered sample loads by which the band width is reduced with benefits to resolution.     

Preparative application of commercial automated gel electrophoresis apparatus to subcellular-sized particles: sequential isolations, fractions re-run, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, yield and purity

The analytical and preparative potential of automated gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path, the HPGE-1000 apparatus (LabIntelligence, Belmont, CA) was further developed in application to subcellular-sized particles. Resolution between two rat liver microsome components in agarose (MetaPhor) gel electrophoresis was found to increase with decreasing agarose concentration to 0.04%. It was less, even in an agarose solution at that low concentration, than that in laterally aggregated 4% polyacrylamide gel. The three components of the microsomal preparation were sequentially isolated from 0.6 and 0.8% agarose gel electropherograms. One fraction when re-electrophoresed was found to exhibit the original mobility and did not give rise to the other components. Yields of each component were near-quantitative after one or two electroelution steps. Based on protein content, no impurities could be detected in two of the microsome fractions; the third fraction contained 2% of nonmicrosome impurity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of all three microsome fractions were indistinguishable from one another and from that of the unfractionated microsome preparation.     

Feasibility of electrophoresis of a subcellular-sized particle in polymer solutions, using automated horizontal gel apparatus

The purpose of this study was to assess the relationship between social adjustment and the risk of depressive relapse during continuation antidepressant treatment. The Social Adjustment Scale-Self Report Version (SAS-SR) was used to assess a broad range of social functioning before and after acute treatment in 41 outpatients with unipolar depression who responded to fluoxetine and then continued on this antidepressant. No differences were detected between those who relapsed and those who stayed well with regard to pre- and posttreatment SAS-SR total or subscale scores. These data suggest that social functioning does not affect the risk of relapse while depressed patients are taking fluoxetine continuation treatment.     

Clinical implications of the types of cryoglobulins determined by two-dimensional polyacrylamide gel electrophoresis

BACKGROUND AND OBJECTIVE: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a new method which can be used to study cryoprecipitates from the sera of cryoglobulinemic patients. It led to the identification of a new type of cryoprecipitate, tentatively named II-III, characterized by polyclonal IgG associated with a mixture of polyclonal and monoclonal IgM. Some discrepancies with the conventional classification of cryoglobulins were revealed. The association of particular clinical features with the classification of cryoglobulins by 2-D PAGE is examined. DESIGN AND METHODS: Sixty consecutive patients affected by cryoglobulinemic syndrome with mixed cryoglobulins were included in the study. All patients were evaluated for cutaneous, articular, hepatic, renal and nervous involvement. The washed cryoprecipitates were typed using both techniques: immunofixation electrophoresis (IFE) and 2-D PAGE. RESULTS: Sixteen (6 cases of type II and 10 of type III by IFE) of 60 cryoprecipitates (26.6%) appeared as type II-III by 2-D PAGE analysis. Nine cases were classified differently by IFE and 2-D PAGE. Mixed cryoglobulins of type II-III were not associated with a particular clinical pattern. Examining the clinical findings in the mono group (those with monoclonal IgM alone) and the poly group (those with polyclonal IgM alone or polyclonal and monoclonal IgM) we found clearly significant differences: more severe liver involvement in the poly group, and higher cryocrit and creatinine values, lower C4 level and more severe purpura in the mono group. INTERPRETATION AND CONCLUSIONS: Our results confirm the reliability of 2-D PAGE in characterizing cryoprecipitates. This sensitive method can demonstrate a higher number of monoclonal components, undetectable by IFE. Type II-III cryoglobulins are not associated with a particular clinical pattern. The presence or absence of polyclonal IgM in mixed cryoglobulins seems to be correlated with some clinical findings.     

Preparative two-dimensional gel electrophoresis of membrane proteins

Electrophoretic techniques, and especially two-dimensional gel electrophoresis (2-DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well as for the identification of protein-protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first or second dimension of 2-DE. Here we describe the modification of a protocol using a combination of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4-dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver-stained gels obtained from identical samples (4 microg). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale-up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialka et al., Electrophoresis 1997, 18, 2582-2590).     

Real-time detection of allele-specific polymerase chain reaction products by automated ultra-thin-layer agarose gel electrophoresis

Ultra-thin-layer agarose gel electrophoresis, a novel combination of agarose slab gel electrophoresis and capillary gel electrophoresis was introduced in conjunction with laser-induced fluorescence (LIF) scanning detection for the analysis of polymerase chain reaction (PCR) products. Allele-specific fragments, amplified from genomic DNA of patients with congenital adrenal hyperplasia (most often caused by mutations of 21-hydroxylase gene, CYP-21), were used as a model system to investigate the applicability, sensitivity and resolving power of the method. The allele-specific products were generated by PCR and separated by ultra-thin-layer agarose gel electrophoresis. The double-stranded DNA fragments were easily visualized in real-time via complexation during the separation process by the intercalator dye TO-PRO-3 which was part of the separation gel-buffer system. In this way, the migrating dsDNA-dye complexes were detected in real-time by a scanning LIF detection system with sub-nanogram sensitivity. The system employs a 632-nm solid-state laser and an avalanche photodiode detector scanning to the separation platform by means of a fiber bundle system. Automated ultra-thin-layer agarose gel electrophoresis with 'on the fly' TO-PRO-3 staining of dsDNA fragments and LIF detection system proved to be a very fast, high-throughput separation method for individual or multiplexed PCR products, with excellent sensitivity.     

Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis

For identification of Rab, Rac, Rho, Ral, Rap, and Arf proteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to high-resolution two-dimensional polyacrylamide gel electrophoresis followed by [alpha-32P]GTP ligand blotting. We have mapped 28 small GTP-binding proteins relative to their isoelectric points and according to their molecular weights and by immunoblotting with specific antibodies. Rab and Rho proteins could be specifically identified by extraction of streptolysin O-permeabilized Madin-Darby canine kidney (MDCK) cells with Rab- and Rho-GDP dissociation inhibitor. We applied the reference mapping to analyze the GTP-binding patterns of synaptosome fractions from rat brain. The purified synaptosomes exhibited specific enrichment of Rab3a, Rab5a, Ral, and several other GTPases. This approach and the map we have produced should provide a useful aid for the analysis of the expression and localization of members of all families of small GTP-binding proteins in various cell types and subcellular fractions.     

Characterization of the major proteins of bovine seminal fluid by two-dimensional polyacrylamide gel electrophoresis

Recently, we demonstrated that the major proteins from bovine seminal plasma BSP-A1, -A2, -A3 and -30-kDa (collectively called BSP proteins) specifically interact with choline phospholipids. These proteins coat the surface of the spermatozoa after ejaculation and are believed to play an important role in membrane modifications occurring during capacitation. In this study we determined the isoelectric point (pl) and analysed the molecular heterogeneity of BSP proteins. Total protein from bovine seminal plasma (CBSP) and purified BSP proteins were iodinated using chloramine T. Samples were reduced, denatured, separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and visualized by autoradiography. Analysis of CBSP proteins demonstrated the presence of polypeptides migrating in the pH range of 3.5-7.8 and at molecular weights (M(r)) between 6 and 100 kDa. isoforms of each BSP protein were found when purified iodinated proteins were analysed by 2D-PAGE. BSP-A1 was found at a M(r) of 16.5 kDa and in the range of pl of 4.7-5.0; BSP-A2 at 16 kDa and at a pl of 4.9-5.2; BSP-A3 at 15 kDa and at a pl of 4.8-5.2, and BSP-30-kDa at 28 kDa and at a pl of 3.9-4.6. Similar results were obtained with immunolocalization of BSP proteins after Western blot using specific antibodies. The treatment of purified iodinated BSP proteins with neuraminidase increased the pl of BSP-30-kDa to 4.8-5.0 and decreased its M(r) to 25 kDa, but no change was observed for BSP-A1, -A2 and -A3. The treatment of BSP proteins with sulfatase or acid phosphatase modified neither their M(r) nor their pl. Furthermore, when CBSP proteins were separated in 2D-PAGE and the gels stained for glycoproteins with dansyl hydrazine, BSP proteins were among the major glycoproteins found in the bovine seminal plasma. In conclusion, BSP proteins are acidic and have several isoforms. Furthermore, the heterogeneity of BSP-30-kDa is mainly due to its sialic acid content.     

Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis

We describe the extraction and enrichment of membrane proteins for separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) after differential solubilization of an Escherichia coli cell lysate. In a simple three-step sequential solubilization protocol applicable for whole cell lysates, membrane proteins are partitioned from other cellular proteins by their insolubility in solutions conventionally used for isoelectric focusing (IEF). As the first step, Tris-base was used to solubilize many cytosolic proteins. The resultant pellet was then subjected to conventional solubilizing solutions (urea, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, dithiothreitol, Tris, carrier ampholytes). Following the completion of this step, 89% of the initial E. coli sample mass was solubilized. Finally, the membrane protein rich pellet was partially solubilized using a combination of urea, thiourea, tributyl phosphine and multiple zwitterionic surfactants. Using N-terminal sequence tagging and peptide mass fingerprinting we have identified 11 membrane proteins from this pellet. Two of these outer membrane proteins (Omp), OmpW and OmpX, have previously been known only as an open reading frame in E. coli, while OmpC, OmpT and OmpTOLC have not previously been identified on a 2-D gel. The prefractionation of an entire cell lysate into multiple fractions, based on solubility, results in simplified protein patterns following 2-D PAGE using broad-range pH 3.5-10 immobilized pH gradients (IPGs). Additional advantages of sample prefractionation are that protein identification and gel matching, for database construction, is a more manageable task, the procedure requires no specialized apparatus, and the sequential extraction is conducted in a single centrifuge tube, minimizing protein loss.     

Determination of Triton X-100 binding to membrane proteins by polyacrylamide gel electrophoresis

The molecular weight of proteins in protein-detergent complexes can be determined from ultracentrifugation experiments if the amount of bound detergent is known. A new sensitive method to measure the binding of the nonionic detergent Triton X-100 to proteins has been developed. For the membrane proteins studied, less than 50 mug of protein was required to achieve an accuracy of 10% in the determination of the detergent-protein weight ratio. The proteins were equilibrated with the detergent by electrophoresis into polyacrylamide gels containing radioactively labelled Triton X-100. The gels were then sliced and the amount of bound detergent calculated from the increase in radio-activity in the slices containing the protein zone. The amounts of protein were determined by amino acid analysis of identical protein zones cut from gels running parallel.     

Genetic relationship between blood and nonblood isolates from bacteremic patients determined by pulsed-field gel electrophoresis

A total of 148 isolates from 55 bacteremic patients were examined by pulsed-field gel electrophoresis. Genetically different nonblood strains were isolated from 13.9% of patients with bacteremia caused by gram-positive cocci and 42.1% with Pseudomonas aeruginosa bacteremia, indicating that antibiograms of a single nonblood P. aeruginosa isolate are not always informative for treatment of bacteremia.     

Alzheimer brain proteins investigated by two-dimensional gel electrophoresis with immobilized pH gradients in the first dimension

Two-dimensional (2-D) gel electrophoresis with immobilized pH gradient 4-8 in the first dimension was applied in the analysis of Alzheimer's disease brain proteins. The silver-stained 2-D maps of extracts from the frontal cerebral cortex were examined. About 800 and 550 protein spots could be observed on the electrophoretograms from the total and buffer-soluble fractions, respectively. In comparing the gels, four protein spots could now be detected which had either been hitherto undetectable (one spot) or which were weaker (two spots) or stronger (one spot) in density (in the controls) [corrected].     

Two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins

A two-dimensional polyacrylamide gel electrophoresis map of bull seminal plasma proteins has been established. About 250 spots were detected after silver staining and polypeptides from 24 spots have been N-terminally sequenced. Major proteins already described in bull seminal plasma, like PDC-109 and aSFP, have been located on the map; proteins not yet reported in male reproductive tracts have been evidenced; for some polypeptides showing a previously unknown N-terminal sequence, structural similarities with proteins described in other organisms have been found. A reference map of seminal plasma proteins could be useful in relating protein pattern changes to physiopathological events influencing the reproductive sphere.     

Agarose gel electrophoresis of cerebrospinal fluid proteins and analysis of the pherogram profiles by analog computer

A micro-method of agarose gel electrophoresis requiring 1--3 ml of cerebrospinal fluid for quantitative analysis of cerebrospinal fluid proteins is described. After concentration of CSF to about 50 microliter by ultrafiltration and refractometric determination of protein, approximately 20--40 microgram of total protein are used for electrophoresis. Photometric scanning of the electrophoretic pattern at two different wavelengths permits quantitative evaluation. The pherograms are analysed by means of a modified DU-PONT analog computer. Factors which influence quantitative electrophoresis are examined. In cerebrospinal fluid of normal children 15 protein fractions are demonstrated: 2 prealbumins, albumin, 5 alpha-, 3 beta- and 4 gamma-globulins.     

Two-dimensional gel electrophoresis of actin-binding proteins isolated by affinity chromatography from human skeletal muscle

Obstructive azoospermia represents approximately 10 % of cases of male hypofertility. It is classified according to the volume of ejaculate. When the latter is normal a proximal obstruction is suspected. Scrotal sonography can help to detect dilation of the epididymal head when clinical findings are equivocal. Ejaculatory duct obstruction (EDO) is suspected when the volume of ejaculate is low. The use of transrectal ultrasonography (TRUS) plays a major role in the investigation of these patients, and endorectal MRI is a very useful adjunct in selected cases. The most common cause of EDO is congenital bilateral absence of vas deferens, which is now thought to be a genital form of cystic fibrosis in 80 % of cases. Consequently, a definitive diagnosis must be made before any attempt at in vitro fertilization. TRUS accurately visualizes abnormalities of the caudal junction of the vas deferens and seminal vesicles, yielding a definitive diagnosis without scrototomy. Other causes of EDO are congenital cysts compressing the distal part of the ejaculatory ducts and inflammatory distal stenosis. The former are accurately identified by TRUS, but the latter give more or less marked signs of obstruction which are only of value in azoospermic patients with a low-volume ejaculate. More invasive imaging is required to diagnose partial obstruction of the ED. Surgical vasography is still the reference, but puncture of the seminal vesicles under TRUS guidance is an attractive alternative, as it permits aspiration of seminal fluid (to seek motile sperm) and vasography without scrototomy. Lastly, endorectal MRI well assesses the relationships between the proximal prostatic urethra and the posterior wall of the ejaculatory ducts, which need to be precisely known when endoscopic resection of the ejaculatory ducts is planned.