I. Introduction of mouse cell therapy for RNase A
RNase A is followed by immunocytotherapy as a method that allows the display of localization to involve RNase-sensitive structures of cellular proteins. This technique is highly dependent on the quality of RNase A used in our laboratory, and our treatment with RNase A in mouse cells showed that heterochromatin 1 (HP1) is rich in heterochromatin between arms. The set depends on the presence of an RNA component.
Second, materials and reagents
CSK buffer: (stored at -20 ° C)
10m M tube (pH 7.0)
100 mM sodium chloride
300 MM cane sugar
3 mM magnesium chloride
Third, the experimental steps
1. The RNase preparation is derived from bovine pancreatic ribonuclease A, an endoribonase that specifically cleaves single stranted RNA. The enzyme attacks the 3-phosphate group of the pyrimidine nucleotide and the cleaved 5-phosphate-linked neighboring nucleotide. RNase A, which works in the presence of cofactors and divalent cations, can be inhibited by RNase inhibitors. This protocol is crucial for the selection and preparation of RNase A. Some companies with RNase A are fully suitable for molecular biology applications, but it can't be used by cells, it just kills them.
2. Dissolve RNase A at a concentration of 10 mg/ml in 0.01 M sodium acetate (pH 5.2); heat to 100 ° C for 15 minutes; allow slow cooling to room temperature; by adding 0.1 volume of 1 M TrisHCl (pH 7. 4), adjust the pH; dispense to the store at -20 ° C
3. Cell extraction
In order to detect the interaction of RNA-dependent proteins in the nucleus, the cells are first extracted with detergent. This step can be removed leaving a soluble protein that binds the protein more tightly.
4. Cover slips grown directly to adherent cells
Wash for 3 minutes at room temperature with 1x PBS; wash with CSK, buffer for 3 minutes at room temperature; incubate in CSK buffer + 0.5% Triton X-100 for 5 minutes at room temperature; CSK buffer for 3 minutes, Wash at room temperature; wash twice with PBS for 3 minutes at room temperature.
5. RNaseA processing
For RNase A-treated Triton X-100 permeable cells (RNase concentration, time and temperature), titration experiments were performed to determine optimal conditions using RNase A Roche. Obvious conditions are used to make adjustments, which not only depend on the RNase, but also on the type of cell being treated. Add 1 mg/ml RNase A to PBS for 10 minutes at room temperature; wash twice with PBS for 3 minutes at room temperature; fix 2% paraformaldehyde in PBS for 15 minutes at room temperature; then proceed dyeing. Specific RNase digestion of known RNA-dependent markers, such as nucleolar fibers, disperses post-processing related proteins for the maturation of ribosomal RNA, which has been monitored. However, several nuclear components (nuclear membrane proteins, lamin B; splicing factor é’ª-35; centromere proteins CENP-A, B, and C) that have not been completely screwed into the nucleus are retained.
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