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PubMed1995

Use of an SDS-gel-separated protein band as a ligand for affinity chromatography: procedure and application to the purification of domain-specific antibodies against alpha-actinin.

This paper describes a simple and efficient method for preparing affinity columns. We used protein separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis as a ligand. Protein bands detected in a polyacrylamide gel were electrophoretically transferred to CNBr-activated Sepharose using a buffer containing Nonidet P-40. The amount of ligand protein coupled to activated Sepharose by our method was almost comparable to that obtained by conventional coupling procedure with a native ligand protein. Using affinity columns prepared by this method, we have successfully purified anti-alpha-actinin antibody and antibodies highly specific to the rod domain of alpha-actinin from the antiserum. This new method should be useful for separating a specific antibody from an antiserum that has been raised against multiple antigens. In addition, the use of the SDS-gel-fractionated band facilitates the coupling of proteins that have low solubility under the coupling conditions.

Actinin↗
PubMed1995

High salt- and SDS-stable DNA binding protein complexes with ATPase and protein kinase activity retained in chromatin-depleted nuclei.

Cell lysis in presence of SDS and proteinase K followed by salting-out of residual polypeptides by dehydration and precipitation with saturated sodium chloride solution [Miller, S.A., Dykes, D.D. and Polesky, H.F., Nucleic Acids Res., 16, 1215, 1988] efficiently resolves deproteinized DNA. However, this DNA is still associated with prominent polypeptides which remain stably attached to DNA during further treatments, e.g. during repeated salting-out steps, prolonged incubation of DNA in 1% SDS or 4 M urea at 56 degrees C and ethanol precipitation. The persistent polypeptides (62, 52 and 40 kDa) released from Ehrlich ascites cell DNA were further characterized. Microsequencing indicates that the DNA binding polypeptides are not yet characterized at the sequence level. Nuclease digestion of the DNA releases stable DNA-protein complexes with the shape of globular particles (12.8 +/- 0.8 nm) and their larger aggregates in which DNA remains protected from nuclease digestion. The isolated DNA-polypeptide complexes show ATPase (Km = 7.4 x 10(-4) M) and protein kinase activity. Antibodies reveal a parallel distribution of the complexes with chromatin, however, the complexes are retained in chromatin-depleted nuclei.

Adenosine Triphosphatases↗
PubMed1983

Isolation of the alpha and beta subunits of canine (Na+,K+)ATPase by using SDS-PAGE and lectin-Sepharose.

(Na+,K+)ATPase from dog kidney was solubilized and denatured by SDS treatment, then applied to a Con A- and WGA-Sepharose column. While the alpha subunit of the ATPase had no affinity for either of the lectin-Sepharoses, the beta subunit specifically bound to WGA-Sepharose and was eluted with N-acetylglucosamine. This property was utilized for the isolation of the alpha and beta subunits by using lectin-Sepharoses and SDS-polyacrylamide gel electrophoresis. The amino acid composition of the alpha subunit thus isolated was in reasonable agreement with the data reported by Kyte (Kyte, J. (1972) J. Biol. Chem. 247, 7642-7649). The amino acid and carbohydrate compositions of the beta subunit were, however, different from his data. The beta subunit contained little histidine (0.1 mol/100 mol amino acid) and a very large amount of carbohydrates (33%). The antibody raised against alpha or beta subunit reacted specifically with the corresponding subunit and with protease-fragmented alpha subunit and neuraminidase-treated beta subunit, respectively, but no cross-reactivity was observed between the two subunits. These results indicate that our alpha and beta subunits were highly purified.

Amino Acids↗
PubMed1992

Guinea pig or rabbit lung flavin-containing monooxygenases with distinct mobilities in SDS-PAGE are allelic variants that differ at only two positions.

Both guinea pig and rabbit express two variants of the 'lung' flavin-containing monooxygenase (FMO), observed as three distinct phenotypes based on mobility differences in SDS-PAGE. Samples of messenger RNA prepared from lungs of the two homozygous phenotypes of the guinea pig were used for the construction of two cDNA libraries. The libraries were screened with a cDNA encoding the rabbit lung FMO, and positive clones for each guinea pig lung FMO variant were isolated and sequenced. A full length clone from each library was found to encode a protein of 535 amino acids containing two pyrophosphate binding sites. Comparison of the sequences of the guinea pig and rabbit lung FMOs shows that their primary structures are 86% identical. The coding region sequences of the guinea pig variants differ at only two positions, and both differences result in amino acid substitutions. Sequence analysis has also been completed on a partially characterized variant of the rabbit lung FMO. As with the guinea pig, the nucleotide and amino acid sequences of the rabbit variants differ at only two positions. The cDNAs encoding the guinea pig variants were expressed in yeast. The activities of the enzymes are characteristic of the lung FMO, and the mobilities of the expressed enzymes are the same as those observed for the variants present in guinea pig pulmonary microsomal preparations. Similar to findings for the rabbit, analysis of genomic DNA indicates that the guinea pig lung FMO is associated with a single gene. The results of cDNA sequence analysis, expression in yeast, and analysis of genomic DNA indicate that the multiple lung FMOs in guinea pig and rabbit are allelic variants whose mobilities in SDS-PAGE are markedly altered by minimal changes in primary structure.

Alleles↗
PubMed1989

An analysis of the antibody response against West Nile virus E protein purified by SDS-PAGE indicates that this protein does not contain sequential epitopes for efficient induction of neutralizing antibodies.

The large envelope (E) protein of flaviruses is the viral surface protein that contains neutralizing epitopes. We have analysed the E protein of the West Nile (WN) flavivirus for neutralizing epitopes generated from linear segments of the protein; if effective, these might allow the synthesis of peptides suitable for vaccination. For this study we used the E protein and defined fragments thereof as antigens in rabbits. The sera thus obtained, containing antibodies to E protein as shown by Western blot analyses, were tested for neutralizing activity by the plaque reduction neutralization test. If the E protein used as antigen was reduced (the native E protein contains six disulphide bridges) and denatured the resulting antibodies did not consistently demonstrate neutralizing activity. These results show that the E protein of WN virus does not contain a linear segment that is able to induce neutralizing antibodies efficiently. Studies using denatured E protein fragments containing subsets of the intact disulphide bridges showed that the local covalent primary structure of the protein involved in each of the six bridges was also insufficient for inducing the synthesis of neutralizing antibodies. The complete E protein, with all six disulphide bridges intact, purified by preparative SDS-PAGE under the conditions used in our experiments could, however, induce the synthesis of neutralizing antibodies in rabbits. These data indicate that at least one complex epitope which is able to induce neutralizing antibodies is not completely denatured or can be reformed to some extent if the complete E protein has been subjected to SDS-PAGE without prior destruction of the disulphide bridges.

Amino Acid Sequence↗
PubMed1987

Electrophoretic analysis of proteins from Mycoplasma hominis strains detected by SDS-PAGE, two-dimensional gel electrophoresis and immunoblotting.

The proteins of 14 strains of Mycoplasma hominis were compared by SDS-PAGE in gradient gels, by two-dimensional (2D) gel electrophoresis of extracts of 35S-labelled cells and by immunoblot analysis of cell proteins. The strains examined included the M. hominis type strain PG21 and 13 others isolated variously from genital tract, mouth, blood, upper urinary tract and a wound. These 14 strains shared 76-99% of proteins in SDS-gradient gel analysis and 41-72% in the 2D gels. As expected, the immunoblot analysis likewise revealed the existence of an extensive common protein pattern in M. hominis, in addition to a number of antigens shared only by some strains.

Antigens, Bacterial↗
PubMed1989

Analysis of the axial filaments of Treponema hyodysenteriae by SDS-PAGE and immunoblotting.

Purified axial filaments from eight serotypes of Treponema hyodysenteriae and two non-pathogenic intestinal spirochaetes were characterized by SDS-PAGE and Western blotting. Axial filaments of all ten strains had similar SDS-PAGE profiles; five major axial filament polypeptides were identified, with molecular masses of 43.8, 38, 34.8, 32.8 and 29.4 kDa. Hyperimmune gnotobiotic pig serum raised against purified axial filaments of strain P18A (serotype 4) cross-reacted with all other serotypes and with the non-pathogens, and convalescent serum taken from a pig with persistent swine dysentery also showed a strong response to the axial filament polypeptides. Hyperimmune gnotobiotic pig serum raised against axial filaments failed to agglutinate viable organisms and did not inhibit growth in vitro. Hence, the axial filaments of T. hyodysenteriae have been identified as major immunodominant antigens, although the role that antibodies to these antigens play in protection has yet to be established.

Animals↗
PubMed1991

Differentiation of Coxiella burnetii isolates by analysis of restriction-endonuclease-digested DNA separated by SDS-PAGE.

Thirty-two isolates of Coxiella burnetii collected from various hosts ranging from arthropods to man were compared by restriction endonuclease (RE) digestion patterns of chromosomal DNA using SDS-PAGE. SDS-PAGE provided better DNA fragment separation than agarose gel electrophoresis and enabled the differentiation of these isolates into six distinct groups on the basis of DNA restriction fingerprints. Two groups of chronic disease isolates could be distinguished, each having unique RE digestion patterns of chromosomal DNA. Three similar but distinct RE digestion patterns were seen among the group of acute disease isolates. Three additional isolates included in this study exhibited a unique RE digestion pattern and also had a unique plasmid type, designated QpDG. DNA-DNA hybridization on selected isolates quantified the relatedness between several groups and supported the classification of these groups as distinct strains.

Animals↗
PubMed1992

Molecular analysis of isolates of Streptococcus suis capsular type 2 by restriction-endonuclease-digested DNA separated on SDS-PAGE and by hybridization with an rDNA probe.

This study was undertaken to assess the discriminatory value of restriction endonuclease (RE) digestion patterns of Streptococcus suis chromosomal DNA using polyacrylamide gel electrophoresis (SDS-PAGE) and DNA-rDNA hybridization. For the RE digestion patterns, DNAs were digested separately with the enzymes BamHI and BglII and the resultant fragments were separated by SDS-PAGE. An Escherichia coli rDNA probe derived from pKK3535 was used for the hybridization. Twenty-three S. suis capsular type 2 isolates recovered from diseased and clinically healthy pigs, from a human case, and from a cow were compared in this study. The majority of isolates associated with septicaemia belonged to one restriction endonuclease analysis (REA) profile group. Isolates associated with pneumonia belonged either to the REA profile group of isolates associated with septicaemia or to a second REA profile group. The REA profiles of isolates from clinically healthy animals were more heterogeneous. The REA profile of the type 2 reference strain, S735, which was originally isolated from a pig, was very different from those of the porcine and bovine isolates but similar to the profile of the human isolate. The profiles obtained after rDNA hybridization were more homogeneous. Although different patterns were detected in the 23 isolates, there was no correlation between the source of the isolate and the patterns observed with this technique.

Animals↗
PubMed1997

Identification of novel species-specific antigens of Mycoplasma hyopneumoniae by preparative SDS-PAGE ELISA profiling.

Mycoplasma hyopneumoniae, M. hyorhinis and M. flocculare are commonly isolated from the respiratory tract of pigs and are phylogenetically related. The identification and characterization of antigens specific for M. hyopneumoniae is crucial for the development of serological reagents and for understanding the mechanisms of pathogenicity of this pathogen. Protein and antigen profiles of six strains of M. hyopneumoniae, four strains of M. hyorhinis and a type strain of M. flocculare were compared using SDS-PAGE and immunoblotting. Five strains of M. hyopneumoniae originally isolated from diverse geographical regions produced similar protein and antigen profiles. One strain, C1735/2, produced a unique protein profile and was poorly immunoreactive, suggesting that some strains of M. hyopneumoniae may possess a structurally modified repertoire of antigens. Major M. hyopneumoniae antigens with molecular masses of approximately 36, 43, 48, 52, 76, 78, 80, 82, 94, 106, 114 and 200 kDa were identified by immunoblotting using hyperimmune pig sera raised against both high and low passage strains of M. hyopneumoniae. Porcine hyperimmune sera raised against the GDL type strain of M. hyorhinis reacted strongly with all M. hyorhinis strains although the profiles displayed considerable variation. Major antigens of molecular mass 42, 49, 52, 78, 80 and 82 kDa were identified in type strains GDL and BTS-7 and field strain 2; however, field strain 1 produced a unique profile. A preparative SDS-PAGE profiling (PPP) technique was developed which enabled quantification of the immunoreactivity of denatured antigens with porcine serum by ELISA. PPP facilitated the rapid identification of species-specific and cross-reactive antigens among the three mycoplasma species. PPP studies revealed several strongly immunoreactive M. hyopneumoniae-specific antigens of 43, 76, 94, 114 and 200 kDa as well as antigens of molecular mass between 52 and 62 kDa which were not apparent in immunoblotting studies. Rabbit monospecific anti-42 kDa serum reacted specifically with a 43 kDa antigen in whole cell lysates of geographically diverse strains of M. hyopneumoniae and failed to cross-react with M. flocculare or M. hyorhinis whole cell lysates. This study has identified a number of M. hyopneumoniae-specific antigens which warrant further investigation to determine their potential as diagnostic reagents and the role they play, if any, in pathogenicity.

Animals↗
PubMed1993

Characterisation of Staphylococcus intermedius isolates from canine pyoderma and from healthy carriers by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis.

Ten Staphylococcus intermedius isolates from cases of canine pyoderma and 10 from healthy carriers were examined by SDS-PAGE of exoproteins, immunoblotting and restriction endonuclease digest analysis. Similarities between banding patterns of the isolates were calculated as Dice coefficients for all three methods. For SDS-PAGE and immunoblotting, no significant differentiation was found between the pyoderma and "healthy" groups. Analysis of DNA digested with BglII indicated that S. intermedius is genetically heterogeneous; Dice coefficients for the pyoderma group were distinct from those for the healthy group (p < 0.001), and cluster analysis confirmed that the pyoderma isolates (9) formed a group separate from the majority (6 of 9) of the normal isolates.

Animals↗
PubMed1994

Application of pyrolysis mass spectroscopy and SDS-PAGE in the study of the epidemiology of Pseudomonas cepacia in cystic fibrosis.

Representative isolates of Pseudomonas cepacia from 15 cystic fibrosis (CF) patients attending the Respiratory Unit of Alder Hey Childrens' Hospital were investigated by SDS-PAGE of whole-cell polypeptides and by pyrolysis mass spectroscopy (PMS). SDS-PAGE was less discriminatory than PMS. Eleven isolates were indistinguishable by PMS and considered to represent re-isolates of an endemic strain; four isolates were distinct from this group, and from one another. P. cepacia was first isolated on the unit in July 1989 from a patient who had attended a UK selection meeting for a Canadian CF camp. A ward and outpatient segregation policy was introduced, but colonisation of further patients occurred. In August 1991, the Adult CF Association recommended that all social activities involving colonised patients should cease. This, and an increased awareness amongst older CF patients of the risks of person-to-person transmission, was associated with a marked decline in new cases. Social activity and hospital admissions were compared for colonised patients during the year before colonisation with P. cepacia, and matched patients who did not acquire the endemic strain. This showed a significantly higher attendance at CF social events for colonised patients, but no significant association between colonisation and hospital admission. These results are strong indirect evidence that transmission of P. cepacia occurs through social contact outside the hospital environment.

Adolescent↗
PubMed1982

Characterization of structural properties for morphological differentiation of melanosomes: II. Electron microscopic and SDS-PAGE comparison of melanosomal matrix proteins in B16 and Harding Passey melanomas.

This study compared the qualitative and quantitative differences of the melanosomal matrix proteins between the ellipsoidal-lamellar melanosomes of B16 melanoma and the spherical-granular melanosomes of Harding Passey (HP) melanoma by electron microscopy and SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis). The purity of the melanosomes at each step of the sample preparations was assayed by % distribution of cytoplasmic marker enzymes and proteins. Characterization of the melanosomal matrix was carried out in material purified by the 2 steps of sucrose density gradient ultracentrifugation and membrane dissociation with BRIJ-35. Eighty-three percent of the matrix proteins were solubilized from both B16 and HP by guanidine HCl (GH), which also caused marked ultrastructural changes in different ways. The inner matrix of HP completely disintegrated into fine grains whereas the matrix of B16 showed a great loss in electron density and was degraded, though its basic framework remained unchanged. Under SDS-PAGE, the melanosomal matrix was dissociated into 14 polypeptide bands with respect to size and charge density, 10 of which (63-85% of total proteins solubilized by GH) were common to both B16 and HP. A significant quantitative difference was noted in the relative amounts of these common proteins. The remaining 4 were unique in molecular weight to each form of melanosome though their relative amounts were low. It is suggested that these chemical differences, i.e., quantitative and qualitative, of the matrix proteins are responsible for the ultrastructural differentiation of melanosomes.

Animals↗
PubMed1995

Auxiliary method for clonal identification of Staphylococcus aureus by protein band pattern of released proteins on SDS-polyacrylamide gel.

A supportive method for clonal identification of Staphylococcus aureus strains was devised. Culture supernatant obtained by cellophane surface culture was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without performing any concentration procedure prior to electrophoresis. The combined use of cellophane surface culture and SDS-PAGE was convenient for determining whether the strains belonged to the same clone or not when conducted in conjunction with other tests for bacteriological characterization.

Bacterial Proteins↗
PubMed1990

A rapid, semi-automated SDS-PAGE identification system for oral anaerobic bacteria.

SDS-polyacrylamide gel electrophoresis is a useful technique in bacterial differentiation and identification. A rapid, semi-automated SDS-PAGE system (Phast System) was assessed for identification of formate-fumarate-requiring, asaccharolytic, Gram-negative oral anaerobes. The system permitted loading, separation and staining of gels within 2 h. Percentage similarities between strains were determined using correlation coefficients and cluster analysis. The protein profiles were sufficiently reproducible provided that distorted profiles were disregarded. Strains were successfully separated into their species, with the exception of Bacteroides ureolyticus NCTC 10939, which appeared to be distinct from other strains of that species. Twenty-nine unidentified formate-fumarate-requiring, sub-gingival plaque strains were suitably clustered with the standard strains as verified by a series of physiological and biochemical tests.

Bacterial Proteins↗
PubMed2005

Oxidant and SDS-stable alkaline protease from a halo-tolerant Bacillus clausii I-52: enhanced production and simple purification.

AIMS: An investigation was carried out on the enhancement of protease production and simple purification of an oxidant and SDS-stable alkaline protease produced by Bacillus clausii I-52 of industrial significance. METHODS AND RESULTS: The supplementation with 0.4% (w/v) NaCl and 0.05% (w/v) FeSO4.7H2O in a culture medium caused an increase in the protease production. The enzyme was purified to homogeneity with overall recovery of 79% and 10-fold purification from culture supernatant using Diaion HPA75, phenyl-Sepharose and DEAE-Sepharose column chromatographies. The protease was a halo-tolerant enzyme with apparent molecular mass of 28 kDa, and the Km and kcat values for N-Succinyl-Ala-Ala-Pro-Phe-pNA at 45 degrees C and pH 11.0 were determined to be 83.9 micromol l(-1) and 238.6 s(-1) respectively. CONCLUSIONS: Bacillus clausii I-52 was identified as a halo-tolerant bacterium, and the extracellular alkaline protease produced by B. clausii I-52 also showed extreme halo-tolerance. The enzyme stability towards SDS and H2O2 could be increased by adding NaCl or propylene glycol to the enzyme solution. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline protease secreted by B. clausii I-52 is significant from an industrial perspective because of its stability against surfactants and oxidants as well as its tolerance towards high salinity. These enzymatic properties suggest its suitable application for industrial purposes.

Bacillus↗
PubMed1996

Anomalous mobility of sulfitolysed proteins in SDS-PAGE. Analysis and applications.

Some cysteine-containing proteins upon sulfitolysis have been found to show anomalously retarded SDS-PAGE mobilities in non-reducing gels. These proteins include bovine serum albumin, ovalbumin, aldolase, ribonuclease and a recombinant fusion protein (XA) consisting of a portion of gamma-interferon linked to the A chain of human insulin. This mobility shift has been employed to determine the stability of the sulfonated products and to study the kinetics of the sulfitolysis reaction. Partially sulfonated products of intermediate shifts were observed at 0.01% beta-ME, while 0.05% beta-ME gave a shift characteristic of the completely reduced protein. The undiluted sulfitolysis reagent reacted with XA to give within 1 min a gel shift characteristic of the fully sulfitolysed protein. Its transition stages could be visualized at 15, 30 and 60 min when the reagent was diluted four-fold. In the presence of 8 M urea, the sulfitolysis of BSA was nearly complete at 30 min when the sulfitolysis reagent was used at a dilution of 1:5. However, under the same conditions BSA was predominantly unsulfitolysed in the absence of urea. In order to elucidate the mechanism of sulfonation shift, several derivatives of XA, e.g. performic acid oxidized, alkylated with (a) iodoacetamide and (b) iodoacetate, have been prepared. While the mobility of XASSO3- was sensitive to the presence of beta-ME, all other derivatives moved in a beta-ME-insensitive fashion. Furthermore, while the nonreducing mobilities of the acidic derivatives (-SSO3-, -SO3- and -SCH2CO2-) were anomalously retarded and identical, the mobility of the iodoacetamide derivative was intermediate between the retarded acidic derivatives above and XA below. These studies have suggested a role of the extended conformation of the A chain of insulin in causing a mobility shift of the acidic derivatives in this series. Similar results were observed in an analogous series of derivatives prepared from BSA. Non-denaturing gel filtration analyses of native vs. sulfitolysed samples of serum albumin, ovalbumin and ribonuclease have indicated that the sulfitolysed proteins elute earlier than their native counterparts and appear to be significantly larger than their true molecular weights. Circular dichroism analysis has indicated significant loss in helicity of sulfitolysed BSA. This suggests that the retarded mobility of sulfitolysed proteins seen on SDS-PAGE is likely to be due to an expansion in the hydrodynamic volumes of these proteins, a phenomenon triggered by cleavage of disulfide bonds and further accentuated by the introduction of strongly negatively charged sulfonates.

Animals↗
PubMed1989

Characterization of selected gram-negative oral microorganisms by SDS-PAGE.

SDS-PAGE analysis of bacterial cell proteins was used to examine a diverse group of oral microorganisms. Most of the species examined, including physiologically similar microorganisms, produced distinct protein patterns which are characterized by the distribution of their major protein bands. The SDS-PAGE system described provides a rapid, simple and standardized identification technique to be used in consort with routine taxonomic procedures.

Bacterial Proteins↗