Immunoprecipitation oocytes

Isom I think it is, and our mutagenesis studies are starting to suggest this. We have a new paper coming out in which we have looked at a mutant called pi stop that lacks the intracellular tail (Meadows et al 2001). In oocytes, this mutant associates very poorly with a and has approximately a 10 000-fold lower affinity for a than wild-type pi. It is expressed as efficiently as wild-type pi yet does not associate with a efficiently. In transfected cells only a small portion of the total mutant pi pool can be co-immunoprecipitated with a, even though they are both present in the plasma membrane. 

Immunoprecipitation analysis can be used to determine if a specific protein, for which an antibody is available, becomes associated with a microinjected RNA. Moreover, antibodies against particular RNA modifications (e.g., 5 cap structures or modified bases) can be employed to determine if an injected RNA undergoes a particular posttranscriptional modification following oocyte injection. 

Preparation of oocyte samples and immunoprecipitation. Extracts containing P-labeled RNAs are prepared either from whole oocytes or isolated nuclear and cytoplasmic fractions by homogenization on ice in a suitable buffer (e.g., 40 mM HEPES, pH 7.9, with KOH 100 mM potassium acetate, 8% (v/v) glycerol, 5 mM MgS04, 2 mM EGTA, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5% (v/v) aprotinin, 2 /ig/ml of each leupeptin and pepstatin, and 0.1 U/jul of RNasin) at a ratio of at least 25 pi of buffer per oocyte (or isolated cytoplasm) or as little as 1-4 pi per isolated nucleus. If the antibody recognizes RNA rather than an... 

An example of an RNA injection/immunoprecipitation experiment is shown in Fig. 2. In this experiment, a mixture of radiolabeled snRNAs were coinjected into oocyte nuclei. Oocyte fractionation over time followed by electrophoresis of the RNAs in both the nuclear and cytoplasmic compartments (Fig. 2A) revealed the nucleocytoplasmic distributions of the injected RNAs. The RNAs present in the nuclear fractions were extracted and immunoprecipitated (Fig. 2B and C) using antibodies recognizing either the m G cap (of the precursor snRNAs) or a hypermethylated m G cap (of the mature snRNAs). This and other experiments (Terns and Dahlberg, 1994 Terns et ai, 1995) demonstrated that U3 RNA and other small nucleolar RNAs differ from most RNA polymerase Il-transcribed RNAs in that they are not exported from the nucleus to the cytoplasm. Furthermore, unlike spliceosomal RNAs, which undergo hypermethy-lation of their m G cap structures within the cytoplasm (Mattaj, 1986), small nucleolar RNAs receive their trimethylated cap structures within the cell nucleus. 

Despite the presence of a functional secretory pathway, as demonstrated by the appropriate post-translational modification and targeting of many proteins expressed from injected mRNAs (Colman, 1984), the oocyte shows a very low level of endogenous secretion (Zehavi-Wilner and Lane, 1977). Radiolabeled secretory proteins translated from microinjected mRNAs can therefore often be detected directly by electrophoresis and fluorography of samples of media without the need for immunoprecipitation or other purification techniques. 

Translation products, particularly secreted proteins in samples of medium, from single mRNAs can often be visualized directly on fluorographs of SDS gels. It is normally necessary, however, to immunoprecipitate from oocyte homogenates to... 

Separation of oocyte homogenate into membrane and cytosol fractions before analysis is a relatively simple procedure. It not only can be used to determine the location of cytoplasmic, secretory and integral membrane proteins, but can also provide a degree of concentration and purification that may enable secretory and membrane proteins to be analyzed without the need for immunoprecipitation. 

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