Abstract
Biochemical approaches are powerful tools for investigating mechanisms of biological processes. Here, we describe several biochemical approaches that have been successfully in our laboratory to study the filamentous fungus Neurospora crassa. These approaches include protein extraction and western blot analysis, protein purification using epitope-tagged fusion protein, protein immunoprecipitation (IP) and Chromatin Immunoprecipitation (ChIP) assays. These methods can also be modified for use in other filamentous fungi.
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References
Horowitz NH (1991) Fifty years ago: the Neurospora revolution. Genetics 127: 631–635
Kushnirov VV (2000) Rapid and reliable protein extraction from yeast. Yeast 16:857–860
Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203
Aparicio O, Geisberg JV, Struhl K (2004) Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. Curr Protoc Cell Biol. Chapter 17:Unit 17.7. Wiley
Renart J, Reiser J, Stark GR (1979) Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. PNAS 76:3116–3120
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. PNAS 76:4350–4354
Sambrook J, Fritsch M (1989) Molecular cloning, 2nd edn. Cold Spring Harbor Laboratory Press, USA
Garceau N, Liu Y, Loros JJ, Dunlap JC (1997) Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 89:469–476
Terpe K (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 60:523–533
He Q, Cheng P, He Q, Liu Y (2005) The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex. Genes Dev 19:1518–1531
Howard GC, Kaser MR (2006) Making and using antibodies: a practical handbook. CRC Press, Boca Raton
Ren B, Robert F, Wyrick JJ, Aparicio O, Jennings EG, Simon I, Zeitlinger J, Schreiber J, Hannett N, Kanin E, Volkert TL, Wilson CJ, Bell SP, Young RA (2000) Genome-wide location and function of DNA binding proteins. Science 290:2306–2309
Tollefsbol TO (2004) Epigenetics protocols. In: Methods in molecular biology, vol 287. Humana Press, USA
Cha J, Huang G, Guo J, Liu Y (2007) Posttranslational control of the Neurospora circadian clock. Cold Spring Harb Symp Quant Biol 72:185–191
Heintzen C, Liu Y (2007) The Neurospora crassa circadian clock. Adv Genet 58:25–66
Dunlap JC, Loros JJ (2004) The neuros-pora circadian system. J Biol Rhythms 19:414–424
Cheng P, Yang Y, Heintzen C, Liu Y (2001) Coiled-coil domain-mediated FRQ-FRQ interaction is essential for its circadian clock function in Neurospora. EMBO J 20:101–108
He Q, Liu Y (2005) Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation. Genes Dev 19:2888–2899
Huang G, Chen S, Li S et al (2007) Protein kinase A and casein kinases mediate sequential phosphorylation events in the circadian negative feedback loop. Genes Dev 21:3283–3295
Xing H, Vanderford NL, Sarge KD (2008) The TBP-PP2A mitotic complex bookmarks genes by preventing condensin action. Nat Cell Biol 10:1318–1323
Acknowledgments
This work was supported by grants from National Institutes of Health and Welch Foundation to Yi Liu.
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Guo, J., Huang, G., Cha, J., Liu, Y. (2010). Biochemical Methods Used to Study the Gene Expression and Protein Complexes in the Filamentous Fungus Neurospora crassa . In: Sharon, A. (eds) Molecular and Cell Biology Methods for Fungi. Methods in Molecular Biology, vol 638. Humana Press. https://doi.org/10.1007/978-1-60761-611-5_14
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DOI: https://doi.org/10.1007/978-1-60761-611-5_14
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