Abstract
A new indirect method for fluorescence localization of proteins making use of the avidin-biotin complex is described. We have prepared both a biotin-modified rabbit heavy meromyosin (BHMM) and a biotin-modified antibody to a smooth muscle myosin. After fixation, cells can be treated with either BHMM, which binds to actin, or the biotinyl antibody, which binds to myosin. In a second step the cell are treated with a fluorescent derivative of avidin (Fl-avidin) which binds to the biotinyl proteins and thus indirectly reveals the location of the cellular action or myosin.
Full Text
The Full Text of this article is available as a PDF (1.3 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ash J. F. Purification and characterization of myosin from the clonal rat glial cell strain C-6. J Biol Chem. 1975 May 10;250(9):3560–3566. [PubMed] [Google Scholar]
- Ash J. F., Vogt P. K., Singer S. J. Reversion from transformed to normal phenotype by inhibition of protein synthesis in rat kidney cells infected with a temperature-sensitive mutant of Rous sarcoma virus. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3603–3607. doi: 10.1073/pnas.73.10.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Green N. M. Avidin. Adv Protein Chem. 1975;29:85–133. doi: 10.1016/s0065-3233(08)60411-8. [DOI] [PubMed] [Google Scholar]
- Hartree E. F. Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem. 1972 Aug;48(2):422–427. doi: 10.1016/0003-2697(72)90094-2. [DOI] [PubMed] [Google Scholar]
- Heitzmann H., Richards F. M. Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3537–3541. doi: 10.1073/pnas.71.9.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
- Lazarides E., Weber K. Actin antibody: the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2268–2272. doi: 10.1073/pnas.71.6.2268. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lowey S., Slayter H. S., Weeds A. G., Baker H. Substructure of the myosin molecule. I. Subfragments of myosin by enzymic degradation. J Mol Biol. 1969 May 28;42(1):1–29. doi: 10.1016/0022-2836(69)90483-5. [DOI] [PubMed] [Google Scholar]
- Sanger J. W. Changing patterns of actin localization during cell division. Proc Natl Acad Sci U S A. 1975 May;72(5):1913–1916. doi: 10.1073/pnas.72.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
- Tonomura Y., Appel P., Morales M. On the molecular weight of myosin. II. Biochemistry. 1966 Feb;5(2):515–521. doi: 10.1021/bi00866a017. [DOI] [PubMed] [Google Scholar]
- Wang K., Ash J. F., Singer S. J. Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4483–4486. doi: 10.1073/pnas.72.11.4483. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber K., Groeschel-Stewart U. Antibody to myosin: the specific visualization of myosin-containing filaments in nonmuscle cells. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4561–4564. doi: 10.1073/pnas.71.11.4561. [DOI] [PMC free article] [PubMed] [Google Scholar]