Ribosomes are the site of protein synthesis in cells, and the most abundant RNA-protein complex in cells, which is huge in eukaryotes. In addition to participating in protein synthesis, ribosomes are also related to cell growth, differentiation, cell life span, embryonic development, and cancer occurrence. In fast-growing yeast cells, cells spend most of their energy to produce and assemble mature ribosomes The related components of ribosomal biology gene transcription accounted for more than 60% of the entire cell transcription system.
In addition, through the study of ribosomes, we can also indirectly understand the expression activity of certain genes or the changes in the expression of specific genes in cells under different stress conditions. By binding RNA on ribosomal subunits and polyribosomes, using qRT-PCR technology, you can directly understand the gene expression activity. How to achieve the separation of ribosomes and obtain relevant information has become the key to ribosome research. The following are the relevant procedures and methods for the separation of ribosomes using the density gradient method, which can be referenced by the relevant personnel of ribosome research.
In the preparation process of density gradient liquid, because the layering method produces a point gradient, the separation effect is poor, so the fully linear density gradient solution prepared by Biocomp is used to prepare a fully linear density gradient solution. Add 1/2 volume of 10% sucrose solution to the SW41 centrifuge tube, and then insert a 1/2 volume of 45% sucrose gradient solution from the bottom of the needle (the addition amount of each gradient solution is based on the scale provided by Biocomp), and place it in Biocomp In the gradient preparation instrument, select the corresponding built-in program (the rotor is SW41, the gradient medium is sucrose, and the gradient range is 10-45%) to run, you can directly obtain a 10-45% completely linear sucrose gradient solution.
Regarding the treatment of cell samples, taking cultured Hela cells as an example, first wash twice with PBS (pH 7.4), then add cell lysis buffer, the buffer contains 50mM Tris-HCL, 100mM KCL (or NaCl), 5mM MgCl2, 1mM DTT, 100ug / ml cycloheximide (cycloheximide), 40U / ml RNasin, and 1% NP-40 and 1% sodium deoxycholate (sodium deoxycholate).
After lysing the cells, first pre-centrifuge the cell lysate, centrifuge at 10000-13000 rpm for 10 min at 4 ° C, then take the supernatant and load it into a 10-45% sucrose density gradient solution. 7.5), 5mM MgCl2, 100mM NaCl and 50ug / ml cycloheximide. The separation of ribosomes is mostly based on the different sedimentation coefficients of the components, so the speed zone centrifugation method is used. Place the centrifuge tube in the Beckman ultracentrifuge, rotate at 36000-40000rpm, centrifuge at 4 ℃ for 2-3h, so that the ribosome subunit and polyribosome are separated in the gradient solution.
Collection and drawing after ribosome separation, manual collection is easy to produce layered disturbances, and absorption peaks cannot be drawn. Therefore, the Biocomp automatic density gradient separation system was used for collection, combined with the UV detector, to detect the absorption of the sample at 260nm, and the absorption peak diagram was drawn in real time. The fraction collector is used to collect the samples in separate tubes, and the RNA on each ribosomal subunit or polyribosome can be directly obtained by the centrifuge tube number corresponding to the absorption peak map, and further related research can be carried out by combining qRT-PCR and other technologies.
references:
1. Adam Amsterdam, Kirsten C. Sadler and Kevin Lai et al. 2004 Many Ribosomal Protein Genes Are Cancer Genes in Zebrafish. PLoS Biology 2 (5): e139. Doi: 10.1371 / journal.pbio.0020139
2. Emiliano P. Ricci, Fabrice Mure and Henri Gruffat et al. 2009. Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2. Nucleic Acids Research, 37 (15): 4932-4943
3. Lakshmi Reddy Palam, Thomas D. Baird and Ronald C. Wek. 2011. Phosphorylation of elF2 Facilitates Ribosomal Bypass of an Inhibitory Upstream ORF to Enhance CHOP Translation. The Journal of Biological Chemistry, 286 (13)
4. Marie-Cecile Didiot, Murugan Subramanian and Eric Flatter et al. 2009. Cells Lacking the Fragile X Mental Retardation Protein (FMRP) have Normal RISC Activity but Exhibit Altered Stress Granule Assembly. Molecular Biology of the Cell, 19,428-437
5. Ruiqing Yang, Sergei A. Gaidamakov and Jingwei Xie et al. 2011. La-Related Protein 4 Binds Poly (A), Interacts with the Poly (A) -Binding Protein MLLE Domain via a Variant PAM2w Motif, and Can Promote mRNA Stability. Molecular and Cellular Biology, 31, 542-556
6. Takao Mori, Chiharu Ogasawara and Toshifumi Inada et al. 2010. Dual Functions of Yeast tRNA Ligase in the Unfolded Protein Response: Unconventional Cytoplasmic Splicing of HAC1 Pre-mRNA Is Not Sufficient to Release Translational Attenuation. Molecular Biology of the Cell, 21 , 3722-3734
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