Hi Ranuka,
Which method you choose will depend exactly on what kind of question you're trying to answer - and what your biological material is (yeast? plant? human? cell line? tissue?). Also, when you ask about an "immuno precipitation" method, what exactly do you mean? Are you looking to fractionate the cell compartments and test for presence of Dicer (see below), or to IP Dicer then look for associated proteins?
With regards to your question: depending on your access to fluorescence microscopes, possibly the simplest/most informative method is to do immunofluorescence (IF) using a Dicer antibody; this will allow you to visualise the distribution within the cell and, if you use a panel of cell structure markers (e.g. Dcp1a for P-bodies, TIA1 for stress granules . . . ) you will also be able to refine your localisation to structures within the nucleus/cytoplasm. If you'd like a protocol for this, let me know
Another option would be to do a subcellular fractionation (for example, separating the cytoplasm, nucleoplasm and chromatin using the Mendez and Stillman protocol. see PMID: 11046155) and then blot the fractions for Dicer by western; however, this method will give you less detailed information than IF and is more prone to difficulties arising from imperfect fractionation (for example, a small amount of nuclear contamination in your cytoplasmic fraction, or vice versa. You can monitor such contamination using fraction-specific antibodies; we use GAPDH [cytoplasm[, Cdk6 [nucleoplasm] and H3 [chromatin]). Again, just ask if you'd like a protocol.
A final option, if you're just looking for a very quick answer, is to check out some of the public localization databases online - for example, Protein Atlas (http://www.proteinatlas.org/), which I know has data on Dicer. While these may be limited in species/cell lines/tissues tested, they may give you an idea.
I hope that all helps! Let me know ify ou have any other questions.
Regards,
Dan
Ranuka,
If you want to look at the subcellular localization of an enzyme, immunofluorescence may not be sufficiently sensitive. Since enzymes are often at low levels in cells, you can amplify the intracellular signal by immunostaining using DAB [Bernard et al 2001]. Alternatively, the fluorescent variety of immunostaining is also available (Tyramide Signal Amplification, Thermo Fisher).
Regards,
Mark
1. Bernard MA et al. (2001) Diminished levels of the putative tumor suppressor proteins EXT1 and EXT2 in exostosis chondrocytes. Cell Motil Cytoskeleton 48(2): 149-162.
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