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Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting.

Proceedings of the National Academy of Sciences of the United States of America, 01 Jul 1985, 82(14):4717-4721
https://doi.org/10.1073/pnas.82.14.4717 PMID: 3161075 PMCID: PMC390975

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Abstract 

Monoclonal antibodies that recognize individual polypeptides of the outer arm dyneins of Chlamydomonas flagella were obtained and used to study the structural relationships between the various polypeptides. Immunoblot analysis showed that the gamma heavy chain of 12S dynein and the alpha and beta heavy chains and Mr 69,000 intermediate chain of 18S dynein each contain immunoreactive sites not found in the other dynein chains or in any other axonemal protein. We also used these antibodies to investigate possible structural similarities between dynein polypeptides from Chlamydomonas and phylogenetically distant species. No crossreactivity was observed with antibodies against either the alpha, beta, or gamma heavy chains, demonstrating that each Chlamydomonas heavy chain has structural features distinct from those present in dyneins from the other species tested. However, one antibody against the Mr 69,000 polypeptide recognized an intermediate chain (Mr 76,000) of latent-activity dynein-1 from the sea urchin Tripneustes gratilla. This result provides further evidence that 18S dynein and latent-activity dynein-1 are related. In the course of the above studies, we modified existing procedures to achieve efficient transfer of high molecular weight proteins from NaDodSO4/polyacrylamide gels to nitrocellulose sheets, and to detect small quantities of protein on nitrocellulose. Our modified procedure for staining total protein on nitrocellulose is rapid, inexpensive, and as sensitive as silver-staining of polyacrylamide gels. These methods should be useful to investigators working with small amounts of protein or requiring resolution of closely migrating polypeptides after transfer to nitrocellulose.

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Proc Natl Acad Sci U S A. 1985 Jul; 82(14): 4717–4721.
PMCID: PMC390975
PMID: 3161075

Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting.

S M King, T Otter, and G B Witman
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Abstract

Monoclonal antibodies that recognize individual polypeptides of the outer arm dyneins of Chlamydomonas flagella were obtained and used to study the structural relationships between the various polypeptides. Immunoblot analysis showed that the gamma heavy chain of 12S dynein and the alpha and beta heavy chains and Mr 69,000 intermediate chain of 18S dynein each contain immunoreactive sites not found in the other dynein chains or in any other axonemal protein. We also used these antibodies to investigate possible structural similarities between dynein polypeptides from Chlamydomonas and phylogenetically distant species. No crossreactivity was observed with antibodies against either the alpha, beta, or gamma heavy chains, demonstrating that each Chlamydomonas heavy chain has structural features distinct from those present in dyneins from the other species tested. However, one antibody against the Mr 69,000 polypeptide recognized an intermediate chain (Mr 76,000) of latent-activity dynein-1 from the sea urchin Tripneustes gratilla. This result provides further evidence that 18S dynein and latent-activity dynein-1 are related. In the course of the above studies, we modified existing procedures to achieve efficient transfer of high molecular weight proteins from NaDodSO4/polyacrylamide gels to nitrocellulose sheets, and to detect small quantities of protein on nitrocellulose. Our modified procedure for staining total protein on nitrocellulose is rapid, inexpensive, and as sensitive as silver-staining of polyacrylamide gels. These methods should be useful to investigators working with small amounts of protein or requiring resolution of closely migrating polypeptides after transfer to nitrocellulose.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
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NCI NIH HHS (1)

NIGMS NIH HHS (2)