scholarly journals Characterization of antibodies directed against the Ankrd2 human muscle protein

2009 ◽  
Vol 61 (4) ◽  
pp. 683-691 ◽  
Author(s):  
Snezana Kojic ◽  
Elisa Medeot ◽  
Georgine Faulkner
Keyword(s):  
Monoclonal Antibody ◽  
Epitope Mapping ◽  
Muscle Protein ◽  
Polyclonal Antibodies ◽  
High Specificity ◽  
Human Muscle ◽  
Deletion Mutants ◽  
C Terminus ◽  
Commercial Antibodies

In order to study the function of the Ankrd2 protein, for which commercial antibodies are not available, we report the production and analysis of polyclonal antibodies to full-length Ankrd2 and its C-terminal and N-terminal regions, as well as a monoclonal antibody to the C-terminus of the protein. Epitope mapping making use of recombinant deletion mutants showed that an epitope located in region 323-333 aa of Ankrd2 is detected by the monoclonal antibody. The high specificity of all four anti-Ankrd2 antibodies for recombinant and endogenous Ankrd2 protein is also demon?strated.

Characterization of Conformers of D 1 of Photosystem II Using Site-Directed Antibodies

1990 ◽  
Vol 45 (6) ◽  
pp. 621-626 ◽  
Author(s):  
R. T. Besford ◽  
B. Thomas ◽  
N. S. Huskisson ◽  
G. W. Butcher
Keyword(s):  
Monoclonal Antibody ◽  
Photosystem Ii ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Present Report ◽  
C Terminus ◽  
Sds Electrophoresis

Abstract Antibodies have been raised to synthetic peptides, corresponding to a region in the loop spanning helices 4 and 5 of D 1 protein (Ala 250-Phe 265) and to a region anticipated to be near the C terminus of mature D 1 (His 332-Ala 345). Polyclonal antibodies to the sequence His 332-Ala 345 reacted with a 32 kDa polypeptide in thylakoid preparations identified as D 1 from its resistance (pea) or susceptibility (wheat) to lysine-C degradation. A monoclonal antibody to His 332-Ala 345 reacted preferentially with a faster migrating polypeptide in SDS electrophoresis, a putative conformer of D 1. Polyclonal antibodies to the sequence Ala 250- Phe 265 also reacted with the faster running polypeptide but not with the population of molecules running at 32 kDa. The putative conformer of D 1 from wheat appears to be more resistant than the main D1 population to lysine-C degradation. Peptide analyses by Takahashi et al. [(1988) FEBS Lett, 240, 6 - 8 ] suggest Asn 335-Ala 344 lies at the processed C terminus. The present report provides immunological confirmation that this sequence is retained in mature D 1.

scholarly journals Further characterization of erythrocyte p80 and the membrane protein defect of In(Lu) Lu(a-b-) erythrocytes

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1475-1481
Author(s):  
MJ Telen ◽  
I Rogers ◽  
M Letarte
Keyword(s):  
Monoclonal Antibody ◽  
Membrane Protein ◽  
Concanavalin A ◽  
Polyclonal Antibodies ◽  
Erythrocyte Ghosts ◽  
Con A ◽  
Down Regulation ◽  
Regulation Of Expression ◽  
Variable Effect

We have previously shown that the In(Lu) gene down-regulates expression of an erythrocyte protein antigen identified by murine monoclonal antibody (MoAb) A3D8. In the present study we have examined In(Lu) Lu(a- b-) erythrocytes for expression of additional epitopes on the erythrocyte 80 kilodalton protein (p80) bearing the A3D8 antigen. Using a total of seven additional MoAbs that recognize three epitopes on erythrocyte p80, we have shown that In(Lu) Lu(a-b-) erythrocytes exhibit down-regulation of expression of all three epitopes. In(Lu) erythrocytes also showed a reduction in their reactivity to rabbit antibodies produced against purified p80 from either erythrocytes or lymphocytes. Furthermore the reactivity of the MoAbs was not altered by treatment of the cells with neuraminidase but was substantially reduced by treatment of cells with trypsin or chymotrypsin. The polyclonal anti- p80 sera were shown to react with a fragment of 50,000 daltons, still associated with erythrocyte ghosts, following treatment of the cells with trypsin or chymotrypsin. Treatment of erythrocytes with the thiol- reactive reagent AET decreased their reactivity with the MoAbs but had a variable effect on their reactivity with polyclonal antibodies. Erythrocyte p80 is a glycoprotein with N-linked oligosaccharides, as demonstrated by its binding to concanavalin A (Con A) and Len culinaris lectins. Following Endoglycosidase F treatment, erythrocyte p80 underwent a reduction in apparent mol wt of 11,000. The presence of a reduced amount of an intact p80 glycoprotein, seen by a decrease in reactivity with MoAbs directed at three distinct epitopes and with two different polyclonal antibodies, suggests that the In(Lu) gene interferes with expression by erythrocytes of the entire p80 glycoprotein.

scholarly journals Generation and characterization of LPA-KIV9, a murine monoclonal antibody binding a single site on apolipoprotein (a)

2020 ◽  
Vol 61 (9) ◽  
pp. 1263-1270
Author(s):  
Ayelet Gonen ◽  
Xiaohong Yang ◽  
Calvin Yeang ◽  
Elena Alekseeva ◽  
Marlys Koschinsky ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Risk Factor ◽  
Peptide Mapping ◽  
Polyclonal Antibodies ◽  
Basic Research ◽  
Murine Monoclonal Antibody ◽  
Therapeutic Approaches ◽  
Apolipoprotein A ◽  
Monoclonal Antibody Binding

Lipoprotein (a) [Lp(a)] is a risk factor for CVD and a target of therapy, but Lp(a) measurements are not globally standardized. Commercially available assays generally use polyclonal antibodies that detect multiple sites within the kringle (K)IV2 repeat region of Lp(a) and may lead to inaccurate assessments of plasma levels. With increasing awareness of Lp(a) as a cardiovascular risk factor and the active clinical development of new potential therapeutic approaches, the broad availability of reagents capable of providing isoform independence of Lp(a) measurements is paramount. To address this issue, we generated a murine monoclonal antibody that binds to only one site on apo(a). A BALB/C mouse was immunized with a truncated version of apo(a) that contained eight total KIV repeats, including only one copy of KIV2. We generated hybridomas, screened them, and successfully produced a KIV2-independent monoclonal antibody, named LPA-KIV9. Using a variety of truncated apo(a) constructs to map its binding site, we found that LPA-KIV9 binds to KIV9 without binding to plasminogen. Fine peptide mapping revealed that LPA-KIV9 bound to the sequence 4076LETPTVV4082 on KIV9. In conclusion, the generation of monoclonal antibody LPA-KIV9 may be a useful reagent in basic research studies and in the clinical application of Lp(a) measurements.

Characterization of cleavage and polyadenylation specificity factor and cloning of its 100-kilodalton subunit

1994 ◽  
Vol 14 (12) ◽  
pp. 8183-8190
Author(s):  
A Jenny ◽  
H P Hauri ◽  
W Keller
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Tryptic Digest ◽  
Cell Extracts ◽  
C Terminus ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor ◽  
Precursor Rna ◽  
Novel Protein

During the formation of the 3' ends of mRNA, the cleavage and polyadenylation specificity factor (CPSF) is required for 3' cleavage of the transcript as well as for subsequent polyadenylation. Using peptide sequences from a tryptic digest, we have cloned the 100-kDa subunit of CPSF. This subunit is a novel protein showing no homology to any known polypeptide in databases. Polyclonal antibodies against the C terminus of the protein inhibit the polyadenylation reaction. Polyclonal and monoclonal antibodies were used to characterize the composition of CPSF. Immunoprecipitations of CPSF from HeLa cell extracts and from labeled chromatographic fractions show the coprecipitation of all four subunits of 160, 100, 73, and 30 kDa. Proteins of 160 and 30 kDa that are specifically cross-linked to precursor RNA by UV irradiation were identified as CPSF subunits by immunoprecipitation. Immunofluorescent detection of CPSF in HeLa cells localized it in the nucleoplasm, excluding cytoplasm and nucleolar structures.

scholarly journals Production, Characterization, and Epitope Mapping of a Monoclonal Antibody against Aspartic Proteinase ofCandida albicans

1999 ◽  
Vol 6 (3) ◽  
pp. 429-433 ◽  
Author(s):  
Byoung-Kuk Na ◽  
Gyung-Tae Chung ◽  
Chul-Yong Song
Keyword(s):  
Monoclonal Antibody ◽  
Candida Albicans ◽  
Candida Tropicalis ◽  
Cathepsin D ◽  
Epitope Mapping ◽  
Candida Parapsilosis ◽  
Aspartic Proteinase ◽  
Ofcandida Albicans ◽  
Human Cathepsin

ABSTRACT A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, andAspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was located in the Asp77 to Gly103 sequence. This antibody could be useful for the characterization of CAP and would be a valuable probe for the detection of CAP antigen in the sera of patients with invasive candidiasis.

scholarly journals Expression and characterization of recombinant trehalose synthase in Bacillus subtilis

2020 ◽  
Vol 42 (4) ◽  
Author(s):  
Nguyen Ngoc Trieu ◽  
Nguyen Thi Quynh ◽  
Nguyen Duc Hoang ◽  
Nguyen Manh Dat ◽  
Tran Duc Long ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Monoclonal Antibody ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Optimal Conditions ◽  
Trehalose Synthase ◽  
Optimal Activity ◽  
His Tag ◽  
C Terminus

Trehalose synthase (TreS, EC 2.4.1.245) is a potential catalyst for synthesis of trehalose, an important natural disaccharide. In this study, the treS gene of Pseudomonas putida (VTCC 12263) was cloned into pHT01 plasmid at BamHI-XbaI position, expressed in Bacillus subtilis (B. subtilis) 1012, and characterized. The recombinant TreS had molecular weight of 68 kDa when fused with 8xHis tag at the C-terminus. catalyzed conversion of maltose to trehalose in optimal conditions had specific activity of 1.664 U/g. Expression of TreS was highest when B. subtilis 1012 harboring pHT01-treS was cultured in TB medium at 30 oC, induced with 1.0 mM IPTG when OD600 reached 0.8 and harvested after 10 hours of induction. The recombinant TreS purified by Ni-sepharose chromatography had specific activity of 41.700 U/g and formed a single band on Western blot with monoclonal antibody against His-tag. The recombinant TreS had optimal activity at 37 oC in 100 mM pH 7.4 PBS and 300 mM maltose. It was inhibited by NaCl, KCl and MgCl2 (retaining 45% or 75% specific activity in buffer containing 5 mM KCl or 5 mM MgCl2, respectively) and stimulated by imidazol (with specific activity increasing by 30–200%).

scholarly journals Structural and Biophysical Characterization of Murine Rif1 C Terminus Reveals High Specificity for DNA Cruciform Structures

2014 ◽  
Vol 289 (20) ◽  
pp. 13903-13911 ◽  
Author(s):  
Rasa Sukackaite ◽  
Malene Ringkjøbing Jensen ◽  
Philippe J. Mas ◽  
Martin Blackledge ◽  
Sara B. Buonomo ◽  
...  
Keyword(s):  
High Specificity ◽  
Biophysical Characterization ◽  
C Terminus

scholarly journals Molecular and Physical Characterization of Burkholderia mallei O Antigens

2002 ◽  
Vol 184 (3) ◽  
pp. 849-852 ◽  
Author(s):  
Mary N. Burtnick ◽  
Paul J. Brett ◽  
Donald E. Woods
Keyword(s):  
Molecular Structure ◽  
Monoclonal Antibody ◽  
Gene Cluster ◽  
Polyclonal Antibodies ◽  
Biosynthetic Gene Cluster ◽  
Physical Characterization ◽  
Burkholderia Mallei ◽  
Biosynthetic Gene ◽  
O Antigens

ABSTRACT Burkholderia mallei lipopolysaccharide (LPS) has been previously shown to cross-react with polyclonal antibodies raised against B. pseudomallei LPS; however, we observed that B. mallei LPS does not react with a monoclonal antibody (Pp-PS-W) specific for B. pseudomallei O polysaccharide (O-PS). In this study, we identified the O-PS biosynthetic gene cluster from B. mallei ATCC 23344 and subsequently characterized the molecular structure of the O-PS produced by this organism.

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