Preparation and Application of Polyclonal and Monoclonal Sequence‐Specific Anti‐Phosphoamino Acid Antibodies

Arlinghaus, Ralph B.

doi:10.1002/0471140864.ps1306s34

Cited by 3 publications

(2 citation statements)

References 36 publications

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“…Monoclonal antibodies (mAbs) have the capacity to distinguish subtle chemical and structural differences in their ligands. This is well illustrated by the ability of phosphopeptide-specific mAbs to recognize their specific ligands, but not the unphosphorylated peptide or a different phosphorylated peptide (34). Our goal in this work was to apply a strategy similar to that used in developing phospho-specific antibodies to generate a mAb specific for K63-linked polyUb that did not cross-react with Ub or other types of Ub linkages.…”

Section: Resultsmentioning

confidence: 99%

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Wang

Matsuzawa

Brown³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Modification of proteins by the addition of lysine (K)-63-linked polyubiquitin (polyUb) chains is suggested to play important roles in a variety of cellular events, including DNA repair, signal transduction, and receptor endocytosis. However, identifying such modifications in living cells is complex and cumbersome. We have generated a monoclonal antibody (mAb) that specifically recognizes K63-linked polyUb, but not any other isopeptide-linked (K6, K11, K27, K29, K33, or K48) polyUb or monoubiquitin. We demonstrate the sensitivity and specificity of this K63Ub-specific mAb to detect K63Ub-modified proteins in cell lysates by Western blotting and in cells by immunofluorescence, and K63Ub-modified TRAF6 and MEKK1 in vitro and ex vivo. This unique mAb will facilitate the analysis of K63-linked polyubiquitylation ex vivo and presents a strategy for the generation of similar reagents against other forms of polyUb.MAP kinase kinase kinase 1 (MEKK1) ͉ TNF receptor-associated factor 6 (TRAF6)

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Section: Resultsmentioning

confidence: 99%

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Wang

Matsuzawa

Brown³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…The peptide used for affinity purification was synthesized by Bio-Synthesis (Lewisville, TX, USA). The peptide was coupled to Affi-Gel 10 (Bio-Rad Laboratories), serum was purified, and the antibody was eluted with 3 M sodium isothiocyanate, and dialysed into phosphate-buffered saline (PBS)/azide according to Sun and Arlinghaus (2004) . An anti-AS antibody was raised against the following peptide: NH 2 -CEHPYLPKHILYRQKE-COOH, which was coupled to keyhole limpet haemocyanin as carrier using the N-terminal cysteine residue.…”

Section: Methodsmentioning

confidence: 99%

Relationship between asparagine metabolism and protein concentration in soybean seed

Pandurangan

Pająk

Molnar

et al. 2012

Journal of Experimental Botany

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The relationship between asparagine metabolism and protein concentration was investigated in soybean seed. Phenotyping of a population of recombinant inbred lines adapted to Illinois confirmed a positive correlation between free asparagine levels in developing seeds and protein concentration at maturity. Analysis of a second population of recombinant inbred lines adapted to Ontario associated the elevated free asparagine trait with two of four quantitative trait loci determining population variation for protein concentration, including a major one on chromosome 20 (linkage group I) which has been reported in multiple populations. In the seed coat, levels of asparagine synthetase were high at 50 mg and progressively declined until 150 mg seed weight, suggesting that nitrogenous assimilates are pre-conditioned at early developmental stages to enable a high concentration of asparagine in the embryo. The levels of asparaginase B1 showed an opposite pattern, being low at 50 mg and progressively increased until 150 mg, coinciding with an active phase of storage reserve accumulation. In a pair of genetically related cultivars, ∼2-fold higher levels of asparaginase B1 protein and activity in seed coat, were associated with high protein concentration, reflecting enhanced flux of nitrogen. Transcript expression analyses attributed this difference to a specific asparaginase gene, ASPGB1a. These results contribute to our understanding of the processes determining protein concentration in soybean seed.

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Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

Zhang

Cao

2012

J Biomed Res

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Recently, serum Golgi protein 73 (GP73) levels have been found to be elevated in patients with hepatocellular carcinoma (HCC), and GP73 has been proposed as a novel marker for HCC. However, GP73 levels in patients remain controversial due to the specificity of the anti-GP73 antibody-based enzyme linked immunosorbent assay (ELISA). Therefore, an anti-GP73 antibody with high specificity was highly demanded. In the present study, by hybridoma screening, we generated an anti-GP73 monoclonal antibody (mAb) designated as 6A2 using recombinant GP73 protein produced by prokaryotic expression. The specificity of 6A2 was evaluated by Western blotting, immunohistochemistry and immunoprecipitation. The results showed that 6A2 recognized GP73 in both native and denatured forms. In addition, we have developed a sandwich ELISA using 6A2 and GP73 polyclonal antibody generated in New Zealand white rabbits according to standard procedures, and measured the serum GP73 level of patients using this assay. Our results showed that serum GP73 levels of HCC patients were significantly higher than those of healthy controls (P = 0.0036). Furthermore, for the first time, GP73 serum level was found to be elevated in patients with breast cancer compared with healthy controls (P = 0.0172).

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Preparation and Application of Polyclonal and Monoclonal Sequence‐Specific Anti‐Phosphoamino Acid Antibodies

Cited by 3 publications

References 36 publications

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Analysis of nondegradative protein ubiquitylation with a monoclonal antibody specific for lysine-63-linked polyubiquitin

Relationship between asparagine metabolism and protein concentration in soybean seed

Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

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