Development and regulation of (1→3,1→4)-β-glucan endohydrolases in germinating wheat (Triticum aestivum)

1993 ◽  
Vol 3 (1) ◽  
pp. 65-73 ◽  
Author(s):  
Doreen M. L. Lai ◽  
Amanda M. Slade ◽  
Geoffrey B. Fincher
Keyword(s):  
Monoclonal Antibody ◽  
Triticum Aestivum ◽  
Monoclonal Antibodies ◽  
Gibberellic Acid ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Apparent Effect ◽  
Glucanase Activity

AbstractThe development of (1→3,1→4)-β-glucan 4-glucanohydrolase (EC 3.2.1.73) has been examined in germinating wheat (Triticum aestivum cv. Millewa) grain, and in isolated aleurone layers and scutella. Activity is first detected in extracts of intact grain 2–3 days after the initiation of germination and thereafter increases until 6 days. In isolated aleurone layers, (1→3,1→4)-β-glucanase activity is secreted for up to 4 days. Treatment of aleurone layers with gibberellic acid (GA3) results in a 2-fold enhancement of secreted enzyme for the first 2 days, but activity decreases after 2 days. (1→3,1→4)-β-Glucanase secretion from GA3-treated aleurone layers clearly precedes the secretion of amylase and (1→4)-β-xylanase. Small but significant levels of cellulase are secreted from aleurone layers after GA3 treatment. Isolated scutella also secrete (1→3,1→4)-β-glucanase but GA3 has little apparent effect on secretion patterns from this tissue. Proteins secreted from excised aleurone layers and scutella, together with those in homogenates of intact, germinated grain, have been examined by Western blot analysis, using monoclonal antibodies against barley (1→3,1→4)-β-glucanases as probes. In each case the wheat (1→3,1→4)-β-glucanase is recognised only by the monoclonal antibody that is specific for barley isoenzyme El.

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

2013 ◽  
Vol 34 (4) ◽  
pp. 257-267 ◽  
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.

Dystrophin in skeletal muscle I. Western blot analysis using a monoclonal antibody

1989 ◽  
Vol 94 (1-3) ◽  
pp. 125-136 ◽  
Author(s):  
Louise V.B. Nicholson ◽  
Keith Davison ◽  
Gavin Falkous ◽  
Colin Harwood ◽  
Elizabeth O'Donnell ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibody ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis

scholarly journals A comparison of antigenic peptides in muscle larvae of severalTrichinellaspecies by two-dimensional western-blot analysis with monoclonal antibodies

Parasite ◽  
2001 ◽  
Vol 8 ◽  
pp. S117-S119 ◽  
Author(s):  
M.A. Dea-Ayuela ◽  
F.M. Ubeira ◽  
A. Pitarch ◽  
C. Gil ◽  
A.R. Martínez-Fernández ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Antigenic Peptides ◽  
Two Dimensional ◽  
Muscle Larvae

Partial characterization of 2-β-mercaptoethanol-soluble surface-associated antigens of Onchocerca volvulus

Parasitology ◽  
1991 ◽  
Vol 102 (3) ◽  
pp. 437-444 ◽  
Author(s):  
F. Engelbrecht ◽  
G. Braun ◽  
V. Connor ◽  
M. Downham ◽  
J. A. Whitworth ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Human Infection ◽  
Onchocerca Volvulus ◽  
Rabbit Serum ◽  
Target Antigen ◽  
Molecular Weights ◽  
Igg1 Monoclonal Antibody

Antigens were extracted from the epicuticle/cuticle of intact female Onchocerca volvulus using 2% 2-β-mercaptoethanol and 1% SDS. In Western blot analysis a human infection serum selected for its high antibody titre against whole worm homogenates did not recognize any component solubilized by 1% SDS. However, the same serum did bind at least 7 antigens among the material extracted with 2-β-mercaptoethanol. These antigens have apparent molecular weights (Mr) of: 15 000, 18 000, 28 000, 78 000, 98 000, 120 000 and 200 000. In ELISA using this preparation as target antigen, 151 out of 153 human infection sera gave positive results. An Onchocerca-specific IgG1 monoclonal antibody, designated Cam1, recognized the 28 000 Mr antigen, which is the most prominent antigen detected by Western blot analysis using human infection sera. In ELISA, using material affinity-purified with Cam1 as target antigen, 149 out of 153 human infection sera gave a positive IgG response. From a cDNA library three expressing clones were isolated with a rabbit serum raised against 2-β-mercaptoethanol solubilized material. One of these clones was recognized by the monoclonal antibody Cam1.

Myc-epitope tagged proteins detected with the 9E10 antibody in immunofluorescence and immunoprecipitation assays but not in Western blot analysis

1998 ◽  
Vol 76 (1) ◽  
pp. 125-128 ◽  
Author(s):  
Huizhou Fan ◽  
Cristy Villegas ◽  
Arthur K Chan ◽  
Jim A Wright
Keyword(s):  
Gene Expression ◽  
Monoclonal Antibody ◽  
Recombinant Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunofluorescence Assay ◽  
Expression Vectors ◽  
Epitope Recognition ◽  
In Cells

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.Key words: recombinant protein, Myc epitope, 9E10, Western blot, gene expression, immunofluorescence assay, immunoprecipitation.

scholarly journals Inhibition of the purified human red-cell Ca2+ pump by a monoclonal antibody

1989 ◽  
Vol 264 (1) ◽  
pp. 87-92 ◽  
Author(s):  
A J Caride ◽  
A Enyedi ◽  
J T Penniston
Keyword(s):  
Monoclonal Antibody ◽  
Steady State ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
State Level ◽  
Red Cell ◽  
Steady State Level ◽  
Calmodulin Binding ◽  
High Affinity

1. A monoclonal antibody (1G4) was raised against the red-cell Ca2+ pump, and it reacted with the pump, as verified by Western blot analysis and by the e.l.i.s.a. method. 2. At 1 mM-ATP and 10 microM-Ca2+, 1G4 inhibited the activity of the purified Ca2+ pump by 40%. 3. Ca2+ pump inhibition by the antibody was non-competitive with regard to Ca2+, calmodulin and the high-affinity portion of the ATP curve. Thus its mechanism was quite different from that of the antibody previously reported [Verbist, Wuytack, Raemaekers, VanLeuven, Cassiman & Casteels (1986) Biochem. J. 240, 633-640], which partially caused inhibition by competition at the ATP site. 4. Antibody 1G4 reduced the steady-state level of phosphorylated intermediate and increased by 50% the calmodulin-activated p-nitrophenyl phosphatase activity of the pump. 5. The experimental results are consistent with the hypothesis that 1G4 inhibits the Ca2+ pump by decreasing the rate of the transition from the E2 form to the E1 form, causing a higher concentration of E2. 6. Analysis by Western blot of the pattern of cross-reaction of 1G4 after tryptic digestion of the pump showed that this antibody reacts with bands of Mr 90,000, 85,000, 50,000 and 33,000. After chymotryptic digestion, the antibody reacts almost exclusively with a fragment of Mr 105,000 that is fully active but is not responsive to calmodulin. Altogether, the results indicate that 1G4 binds to an epitope involved in the functional properties of the enzyme but which is not related to the calmodulin-binding domain.

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