Characterization of a monoclonal antibody against active pectate lyase from Erwinia carotovora

1989 ◽  
Vol 35 (6) ◽  
pp. 651-655 ◽  
Author(s):  
L. J. Ward ◽  
S. H. De Boer
Keyword(s):  
Monoclonal Antibody ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Protein Band ◽  
Major Protein ◽  
Western Blots ◽  
Diverse Range ◽  
Purified Antigen

A monoclonal antibody (2E2) produced against pectate lyase from Erwinia carotovora ssp. carotovora reacted with a 41-and a 44-kilodalton protein on Western blots of concentrated Erwinia culture supernatants resolved by sodium dodecyl sulphate – polyaerylamide gel electrophoresis. It was unequivocally shown that monoclonal 2E2 reacted with an active form of pectate lyase by affinity purifying the antigen with the monoclonal. The affinity-purified antigen was enzymatically active and moved as a single protein band in a nonequilibrium isoelectric focusing gel. Monoclonal 2E2 reacted with the pectate lyases of a diverse range of E. carotovora ssp. carotovora, ssp. atroseptica, and ssp. betavasculorum strains, as well as with one of three strains of E. chrysanthemi. The electrophoretic mobility of the major protein (44 kilodaltons) that reacted with 2E2 was identical within a subspecies but differed among subspecies.Key words: Erwinia carotovora, pectate lyase, monoclonal antibody.

scholarly journals β-Defensin 22 is a major component of the mouse sperm glycocalyx

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 753-765 ◽  
Author(s):  
Ashley I Yudin ◽  
Theodore L Tollner ◽  
Cathy A Treece ◽  
Robert Kays ◽  
Gary N Cherr ◽  
...  
Keyword(s):  
Vas Deferens ◽  
Ultrastructural Level ◽  
Protein Band ◽  
Seminal Vesicles ◽  
Major Site ◽  
Western Blots ◽  
Sperm Surface ◽  
Single Protein ◽  
Cauda Epididymidis ◽  
Purified Antigen

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54–57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54–57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54–57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.

Identification of circulating growth hormone-binding proteins in domestic poultry: an initial characterization

1991 ◽  
Vol 130 (1) ◽  
pp. 115-NP ◽  
Author(s):  
R. Vasilatos-Younken ◽  
B. J. Andersen ◽  
R. W. Rosebrough ◽  
J. P. McMurtry ◽  
W. L. Bacon
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Binding Proteins ◽  
Sodium Dodecyl ◽  
Mouse Serum ◽  
Nucleotide Sequence Analysis ◽  
Major Protein ◽  
Western Blots ◽  
Major Band

ABSTRACT Multiple growth hormone (GH)-binding proteins (GHBPs) were identified in serum and plasma samples from domestic chickens and turkeys. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis on 10% acrylamide, 2·7% bis discontinuous gels under reducing conditions and electrotransferred to nitrocellulose paper. Western blots were incubated with 125I-labelled recombinant chicken GH (cGH) or bovine GH and GHBPs visualized by means of autoradiography. In fresh samples (< 2 h from collection to gel electrophoresis), multiple minor high Mr bands were evident between approximately 72 000 and 175 000. Two major bands were observed at approximately 69 500 and 27 500. The latter is consistent with previous reports for the rat and mouse serum GHBPs based on nucleotide sequence analysis. The minor bands were essentially undetectable after storage at −25 °C for several months, and an additional major band at Mr approximately 52 500 appeared. The Mr-69 500 major protein contained N-linked carbohydrate, as determined by a reduction in molecular size by treatment with peptide N-glycosidase F. Binding of 125I-labelled GH was partially inhibited by co-incubation with 50 μg unlabelled pituitary-derived cGH/ml and excess unlabelled porcine GH as well as ovine prolactin, but not by bovine insulin. Non-specific binding of 125I-labelled GH by serum albumin was also observed. A comparison was made between these GHBPs and the hepatic GH receptor (e.g. molecular weight estimates, affinity for homologous versus heterologous GHs, cross-reactivity with prolactin, presence of N-linked carbohydrate). The origin and relationship among the various molecular weight species of GHBPs identified, and their potential role in regulation of the biological activity of GH in birds, remain to be determined. Journal of Endocrinology (1991) 130, 115–122

scholarly journals Purification to homogeneity of a high molecular weight human B cell growth factor; demonstration of specific binding to activated B cells; and development of a monoclonal antibody to the factor.

1985 ◽  
Vol 162 (4) ◽  
pp. 1319-1335 ◽  
Author(s):  
J L Ambrus ◽  
C H Jurgensen ◽  
E J Brown ◽  
A S Fauci
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
B Cells ◽  
Growth Factor ◽  
Cell Growth ◽  
B Cell ◽  
High Molecular Weight ◽  
Sodium Dodecyl ◽  
Western Blots ◽  
B Cell Growth Factor

High molecular weight B cell growth factor (HMW-BCGF) produced by a T cell line was purified to homogeneity and demonstrated to bind specifically to activated human B cells. A monoclonal antibody to HMW-BCGF was developed that (a) specifically inhibited the activity of HMW-BCGF in enhancing B cell proliferation, (b) specifically bound to HMW-BCGF in Western blots, (c) specifically absorbed HMW-BCGF activity from culture supernatants, and (d) specifically absorbed an internally labeled protein from T-ALL supernatant which comigrates with HMW-BCGF on sodium dodecyl sulfate-polyacrylamide gels. This antibody should help in cloning the gene for HMW-BCGF and further exploring the physiologic roles of HMW-BCGF.

Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

scholarly journals Variable major proteins of Borrellia hermsii.

1982 ◽  
Vol 156 (5) ◽  
pp. 1312-1324 ◽  
Author(s):  
A G Barbour ◽  
S L Tessier ◽  
H G Stoenner
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibodies ◽  
Antigenic Variation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Major Protein ◽  
Relapsing Fever ◽  
Western Blots

Borrelia hermsii, a relapsing fever agent, manifests antigenic variation in vivo and in vitro. We studied three mouse-passaged serotypes of strain HS1 (7, 14, and 21) and a HS1 derivative obtained after multiple in vitro passages (C serotype). All four serotypes had two major proteins in whole cell lysates fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One major protein species (pII) had the same apparent subunit molecular weight (or approximately 3.9 X 10(4) in all the serotypes. In contrast, the other abundant protein in lysates, pI, had a different apparent molecular weight in each serotype. In one gel the molecular weights of pIc, pI7, pI14, and pI21 were 1.9, 4.2, 4.1, and 4.0 X 10(4), respectively. Serotype-specific mouse antisera bound to both hemologous and heterologous pIIs, to homologous pI, but not to heterologous pI in Western blots. Hybridomas were raised from spleens of mice infected with B. hermsii. Monoclonal antibodies were identified by immunofluorescence assays using whole organisms. Monoclonal antibodies specific for serotype 7 (H1826) or for serotype 21 (H3326) bound only to pI7 or pI21, respectively, in Western blots. The surface location of the pI was suggested not only by the immunofluorescence studies but also by the labeling of pI7 and pI21 when whole cells of serotypes 7 and 21 were incubated with 125I in the presence of Iodogen. Under the same circumstances, pII was relatively poorly labeled. These studies have identified the variable pI proteins of B. hermsii as serotype-specific antigens. A change from one pI to another may be the basis of antigenic variation of Borrelia species during relapsing fever.

The effect of ageing cooled milk on the composition of the fat globule membrane

1972 ◽  
Vol 39 (1) ◽  
pp. 95-105 ◽  
Author(s):  
M. Anderson ◽  
G. C. Cheeseman ◽  
Dorothy J. Knight ◽  
W. F. Shipe
Keyword(s):  
Gel Electrophoresis ◽  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Band ◽  
Major Protein ◽  
Fresh Milk ◽  
Major Protein Band ◽  
Fat Globule

SummaryThe effect of ageing fresh milk for 24 h at 4°C on the composition of 4 fractions of milk fat globule membrane (FGM) – microsomal pellet membrane (MPM), deoxycholate soluble membrane (DOCM), high density membrane (HDM) and low density membrane (LDM) – prepared from washed cream treated with sodium deoxycholate (DOC), was studied for milk of individual cows. Total FGM and its fractions were solubilized by treatment with sodium dodecyl sulphate (SDS), EDTA and β-mercaptoethanol, and the dissociated FGM proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS.Ageing resulted in a greater loss of phospholipid during cream preparation than was found with fresh milk, and also in a reduction in the total amount of FGM isolated. Loss of FGM material was confined to MPM, DOCM and LDM and was accompanied by compositional changes in DOCM and LDM, quantitatively the most significant fractions. Ageing also produced changes in the gel electrophoresis patterns of DOCM, where the band of greatest mobility (mol. wt about 16000) was partially lost, and of LDM, where there was an increase observed in the major protein band.The implication of the results on the proposed models of FGM structure is discussed.

scholarly journals Characterization of an EnterotoxigenicEscherichia coli Strain from Africa Expressing a Putative Colonization Factor

1999 ◽  
Vol 67 (8) ◽  
pp. 4019-4026 ◽  
Author(s):  
Sami B. Khalil ◽  
Frederick J. Cassels ◽  
Hind I. Shaheen ◽  
Lewis K. Pannell ◽  
Nemat El-Ghorab ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Coli Strain ◽  
Spectrometric Analysis ◽  
Dot Blot ◽  
Single Protein Band ◽  
Single Protein

ABSTRACT An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H− that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt. This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae. Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M r 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide). A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay. Reactivity was temperature dependent, with cells displaying reactivity when grown at 37°C but not when grown at 22°C. Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band. Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A. These structures were rigid and measured 6.8 to 7.4 nm in diameter. Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da. The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India. Cumulatively, our results suggest that this fimbria is CS19. Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC.

scholarly journals Purification and characterization of OBF1: a Saccharomyces cerevisiae protein that binds to autonomously replicating sequences.

1989 ◽  
Vol 9 (7) ◽  
pp. 2906-2913 ◽  
Author(s):  
S C Francesconi ◽  
S Eisenberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Protein Band ◽  
Binding Activity ◽  
Physical Parameters ◽  
Major Protein ◽  
Dna Binding Activity ◽  
Major Protein Band

We previously identified a protein activity from Saccharomyces cerevisiae, OBF1, that bound specifically to a DNA element present in autonomously replicating sequences ARS120 and ARS121 (S. Eisenberg C. Civalier, and B. K. Tye, Proc. Natl. Acad. Sci. USA 85:743-746, 1988). OBF1 has now been purified to near homogeneity by conventional protein and DNA affinity chromatography. Electrophoresis of the purified protein in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of two polypeptides. The major protein band had a relative molecular size of 123 kilodaltons, and the minor protein band, which constituted only a small fraction of total protein, had a molecular size of 127 kilodaltons. Both polypeptides cochromatographed with the specific ARS120 DNA-binding activity and formed a stable protein-DNA complex, isolatable by sedimentation through sucrose gradients. Using antibodies, we have shown that both polypeptides are associated with the isolated protein-DNA complexes. The ARS DNA-binding activity had a Stokes radius of 54 A (5.4 nm) and a sedimentation coefficient of 4.28S, as determined by gel filtration and sedimentation through glycerol gradients, respectively. These physical parameters, together with the denatured molecular size values, suggested that the proteins exist in solution as asymmetric monomers. Since both polypeptides recognized identical sequences and had similar physical properties, they are probably related. In addition to binding to ARS120, we found that purified OBF1 bounds with equal affinity to ARS121 and with 5- and 10-fold-lower affinity to ARS1 and HMRE, respectively. Furthermore, in the accompanying paper (S. S. Walker, S. C. Francesconi, B. K. Tye, and S. Eisenberg, Mol. Cell. Biol. 9:2914-2921, 1989), we demonstrate the existence of a high, direct correlation between the ability of purify OBF1 to bind to ARS121 and optimal in vivo ARS121 activity as an origin of replication. These findings, taken together, suggest a role for OBF1 in ARS function, presumably at the level of initiation of DNA replication at the ARS.

Cloning and characterization of a fox sperm protein FSA-1

1994 ◽  
Vol 6 (6) ◽  
pp. 761 ◽  
Author(s):  
S Beaton ◽  
A Cleary ◽  
Have J ten ◽  
MP Bradley
Keyword(s):  
Fetal Brain ◽  
Protein Band ◽  
Major Protein ◽  
Structural Protein ◽  
Testicular Germ Cell ◽  
Western Blots ◽  
Sperm Protein ◽  
Major Protein Band ◽  
High Stringency ◽  
Rna Transcript

A monoclonal antibody was raised to a fox sperm protein (FSA-1) which was found to be localized to the inner acrosomal compartment of sperm fixed in methanol. Western blots of testicular germ cell membrane extracts probed with this antibody identified a major protein band with a molecular weight of 36,000. Immunofluorescent studies on fox testis sections showed that the antigen is expressed on round and elongating spermatids on a crescent-shaped structure, which probably represents the developing acrosome. An antibody specific for FSA-1 was used to screen a fox testis cDNA library for its cognate gene. An 875-bp cDNA clone was isolated and sequenced revealing an open reading frame. Searches of the GenBank and EMBL databases with the nucleic acid sequence revealed significant homology (86%) of FSA-1 with 406 bases of an unidentified RNA transcript from human fetal brain (EST02625). Northern blot analysis of fox testis RNA samples identified an RNA transcript of approximately 0.9 kb during the months when spermatogenesis is active. Zoo Northern blots (at high stringency) reveal an RNA transcript of a similar size present in testis RNA from dogs and mice. Zoo Southern analysis (high stringency) reveal genomic sequences present in dogs, mice, cattle and sheep. At present, the function of the FSA-1 gene product remains unknown, but it may play a role as a structural protein component of the acrosome.

scholarly journals Survey and Characterization of Viruses in Sweetpotato from Zimbabwe

Plant Disease ◽  
1997 ◽  
Vol 81 (10) ◽  
pp. 1115-1122 ◽  
Author(s):  
Farayi Chavi ◽  
A. Ian Robertson ◽  
Benedictus J. M. Verduin
Keyword(s):  
Amino Acids ◽  
Coat Protein ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Mottle Virus ◽  
Major Protein ◽  
Degenerate Primers ◽  
Vein Clearing ◽  
Major Protein Band

Thirty-one clones of sweetpotatoes collected from some parts of Zimbabwe were used as inoculum sources to mechanically inoculate 13 experimental hosts: Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Datura stramoniumitalic, Gomphrena globosa, Ipomoea purpurea, I. quamoclit, I. rubrocorulea, Nicotiana benthamiana, N. clevelandii, N. glutinosa, N. rustica, and N. tabacum. Systemic vein clearing was observed in N. benthamiana inoculated with buffered sap from nine clones. Purification of the vein clearing inducing agent from one of the sweetpotato clones gave yields ranging from 2 to 17 mg/kg and the A260nm/A280nm was around 1.2. Electron microscopy revealed flexuous filamentous particles with a modal length of 830 nm. Protein analysis of purified virus preparations by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a major protein band of 40 kDa, and this was assumed to be the viral coat protein. Minor protein bands of 27, 37, and 46 kDa were also observed. The viral protein degraded upon storage at 4°C over time to yield a protein band of 27 kDa. Polyclonal antiserum was produced against the purified virus. Protein A gold labeling of the purified virus incubated with available antisera; sweetpotato chlorotic stunt virus (SPCSV), sweetpotato feathery mottle virus strain russet crack (SPFMV-RC), sweetpotato feathery mottle virus, sweetpotato mild mottle virus, sweetpotato latent virus, sweetpotato chlorotic fleck virus, and sweetpotato caulimo-like virus resulted in a higher labeling density with the antiserum of SPFMV-RC than with the antiserum of SPCSV, while the other sera did not react. Further characterization of the vein clearing inducing agent was attempted by reverse transcription-polymerase chain reaction amplification of total RNA with degenerate primers for potyviruses and an oligo dT primer and PCR products of correct size were obtained. The nucleotide sequence was determined and the amino acid of the polyprotein deduced. Comparison with other strains of SPFMV showed strong similarity except for an insertion of 22 amino acids at the N-terminus of the coat protein. The coat protein size of 335 amino acids is the biggest SPFMV so far determined.

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