Analysis of purified human liver α-l-fucosidase by western-blotting with lectins and polyclonal and monoclonal antibodies

Western-blot analysis [with lectins, polyclonal antibodies (pAbs) and four monoclonal antibodies (mAbs)] was employed to investigate the structural relationship between the separated isoforms and subunits of purified human liver alpha-L-fucosidase. SDS/PAGE and Western-blot analysis indicated the presence of two protein bands of 51 kDa and 56 kDa that were recognized by the pAbs. Polyacrylamide-gel isoelectric focusing (PAG-IEF) followed by blotting indicated that the pAbs and mAbs recognized at least five fucosidase isoforms (pI values 3.6-6.0). Lectin blotting indicated an enrichment of sialic acid residues in the more acidic isoforms. Western-blot analysis indicated that four mAbs recognized the 51 kDa subunit and at least two mAbs recognized the 56 kDa subunit. The subunit composition of the isoforms (separated by PAG-IEF) of human liver alpha-L-fucosidase was investigated by SDS/PAGE. One or two closely spaced bands were found for each isoform with a trend of increasing relative amounts of the high-molecular-mass band in the more acidic isoforms relative to the more neutral isoforms. Neuraminidase treatment of alpha-L-fucosidase resulted in a decrease in the amount of the high-molecular-mass subunit and an increase in the amount of the low-molecular-mass subunit, suggesting that these subunits are related at least in part by sialic acid residues. In addition, blotting with lectins indicated the presence of sialic acid residues only in the high-molecular-mass subunit. N-Glycanase treatment led to the disappearance of the glycosylated 56 kDa and 51 kDa protein bands and the appearance of non-glycosylated protein bands at 48 kDa and 45 kDa. The overall results indicate that (1) N-glycosylation contributes to, but does not account completely for, structural differences in the fucosidase subunits and (2) the more acidic isoforms of fucosidase contain enriched relative amounts of the sialylated high-molecular-mass subunit.

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Spliced mRNA Encoding the Murine Cytomegalovirus Chemokine Homolog Predicts a β Chemokine of Novel Structure

Journal of Virology ◽

10.1128/jvi.73.5.3682-3691.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3682-3691 ◽

Cited By ~ 46

Author(s):

Margaret R. MacDonald ◽

Mary W. Burney ◽

Stuart B. Resnick ◽

Herbert W. Virgin

Keyword(s):

Monoclonal Antibodies ◽

Western Blot ◽

Molecular Mass ◽

Blot Analysis ◽

Western Blot Analysis ◽

Stop Codon ◽

Murine Cytomegalovirus ◽

Fibroblast Cells ◽

Cos Cells ◽

Amino Terminal

ABSTRACT A viral mRNA of the late kinetic class expressed by murine cytomegalovirus (MCMV) contains an open reading frame (ORF) whose predicted protein, designated MCK-1, has homology to β chemokines (M. R. MacDonald, X.-Y. Li, and H. W. Virgin IV, J. Virol. 71:1671–1678, 1997). The present study analyzed further the structure of the transcript in infected fibroblast cells. A splicing event removed the MCK-1 stop codon, bringing a downstream ORF into frame with the chemokine homolog and demonstrating that the MCK-1 ORF was an exon of a larger gene. The predicted 31.4-kDa protein, designated MCK-2, contains a putative amino-terminal signal sequence and a β chemokine domain, followed by a carboxyl-terminal domain without significant homology to known proteins. Quantitative analysis of mRNA forms in MCMV-infected fibroblast cells at late times after infection indicated that the viral chemokine RNA was predominantly spliced. There was no evidence for expression of RNA encoding either MCK-1 or MCK-2 at immediate early or early times after infection with MCMV. Monoclonal antibodies generated against bacterially expressed MCK-2 recognized multiple proteins in the range of ∼30 to ∼45 kDa in Western blot analysis of MCK-2 expressed in transfected COS cells. The monoclonal antibodies immunoprecipitated a similar group of proteins in transfected COS cells metabolically labeled with radioactive cysteine. Radiolabelled protein of apparent higher molecular mass was immunoprecipitated from culture medium overlying the transfected cells, suggesting that posttranslationally modified MCK-2 can be secreted. Two proteins with apparent molecular mass suggestive of posttranslational modification were detected by Western blot analysis of cells harvested at late times after infection with MCMV. These studies show that MCMV encodes and expresses a β chemokine homolog with a novel predicted structure.

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OC125, M11 and OV197 Epitopes Are Not Uniformly Distributed in the Tandem-Repeat Region of CA125 and Require the Entire SEA Domain

Disease Markers ◽

10.1155/2013/917898 ◽

2013 ◽

Vol 34 (4) ◽

pp. 257-267 ◽

Cited By ~ 15

Author(s):

Alessandro Bressan ◽

Francesca Bozzo ◽

Carlo Alberto Maggi ◽

Monica Binaschi

Keyword(s):

Ovarian Cancer ◽

Monoclonal Antibodies ◽

Western Blot ◽

Tandem Repeat ◽

Blot Analysis ◽

Western Blot Analysis ◽

Human Cancer ◽

Ovarian Cancer Cells ◽

Repeat Region ◽

Tandem Repeat Region

The human cancer antigen 125 (CA125) is over-expressed in epithelial ovarian cancer cells and it plays a role in the pathogenesis of ovarian cancer. This protein presents a repeat region containing up to sixty tandem repeat units. The anti-CA125 monoclonal antibodies have been previously classified into three groups: two major families, the OC125-like antibodies and M11-like antibodies, and a third group, the OV197-like antibodies. A model in which a single repeat unit contains all the epitopes for these antibodies has been also proposed, even if their exact position is still undetermined. In the present work, the affinities of the monoclonal antibodies, representative of the three families, have been investigated for different CA125-recombinant repeats through Western blot analysis. Different patterns of antibody recognition for the recombinant repeats show that CA125 epitopes are not uniformly distributed in the tandem repeat region of the protein. The minimal region for the recognition of these antibodies has been also individuated in the SEA domain through the subcloning of deleted sequences of the highly recognized repeat-25 (R-25), their expression as recombinant fragments inE. coliand Western blot analysis. Obtained data have been further confirmed by ELISA using the entire R-25 as coating antigen.

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Identification of the German Cockroach Allergens in Korean Atopy Using SDS - PAGE and Western Blot Analysis

Annals of Dermatology ◽

10.5021/ad.2000.12.4.247 ◽

2000 ◽

Vol 12 (4) ◽

pp. 247

Author(s):

Chun Wook Park ◽

Sang Dong Kim ◽

Cheol Heon Lee ◽

Dong-Kyu Lee

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

German Cockroach ◽

Sds Page

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A rapid microbead method for breaking pathogenic mycobacteria: Application in SDS-PAGE and western blot analysis

Current Microbiology ◽

10.1007/bf01571100 ◽

1992 ◽

Vol 24 (6) ◽

pp. 311-317 ◽

Cited By ~ 2

Author(s):

Nalin Rastogi ◽

Valérie Labrousse ◽

Claude Barreau

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Sds Page

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Outer Membrane Protein (OMP) Profiles of Pasteurella multocida Isolates Associated with Haemorrhagic Septicaemia by SDS-PAGE and Western Blot Analysis

Asian Journal of Animal and Veterinary Advances ◽

10.3923/ajava.2014.513.518 ◽

2014 ◽

Vol 9 (8) ◽

pp. 513-518 ◽

Cited By ~ 1

Author(s):

S.H. Somshekhar ◽

B.M. Veeregowda ◽

V.V.S. Suryanaray ◽

G. Leena ◽

K. Dhama ◽

...

Keyword(s):

Membrane Protein ◽

Western Blot ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Blot Analysis ◽

Western Blot Analysis ◽

Pasteurella Multocida ◽

Haemorrhagic Septicaemia ◽

Sds Page

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Investigations on ornithobacterium rhinotracheale in broiler flocks in elazig province located in the east of turkey

Veterinární Medicína ◽

10.17221/5709-vetmed ◽

2012 ◽

Vol 49 (No. 8) ◽

pp. 305-311 ◽

Cited By ~ 7

Author(s):

G. Ozbey ◽

H. Ongor ◽

D. T Balik ◽

V. Celik ◽

A. Kilic ◽

...

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Sodium Dodecyl ◽

Enzyme Linked Immunosorbent Assay ◽

Rrna Gene ◽

Major Band ◽

Cell Extracts ◽

Sds Page ◽

Ornithobacterium Rhinotracheale

In the present study, lung, trachea and serum samples from broiler flocks slaughtered at an abattoir in Elazig province located in the East of Turkey were examined for the presence of Ornithobacterium rhinotracheale using culture and enzyme-linked immunosorbent assay (ELISA). The identity was latter proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis, and polymerase chain reaction (PCR) assays. A total of 324 serum and 250 lung and trachea samples were collected from 10 commercially reared chicken flocks showing respiratory manifestations. The samples were obtained from different flocks. The causative agent (ORT) was isolated from trachea (1.5%) of five chickens and from both lung and trachea (0.4%) of only one chicken in the bacteriological examination of tissues. The presence of antibodies against ORT was detected in 33 (10.2%) of the 324 sera by ELISA. A 784 bp fragment of the 16S rRNA gene was amplified using specific primers in the PCR. All ORT isolates that were positive by culture were also detected to be positive by the PCR. SDS-PAGE protein profiles of whole cell extracts showed a high similarity for all the isolates with a major band of the molecular weight of 33 kDa (kiloDalton). Results of Western blot analysis indicate four antigenic fractions predominantly with molecular weights of 33, 42, 52 and 66 kDa.

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