Journal: Nucleic Acids Research
Article Title: Effective extraction of polyribosomes exposes gene expression strategies in primary astrocytes
doi: 10.1093/nar/gkad131
Figure Lengend Snippet: Development of an optimized method for efficient extraction of membrane-bound polyribosomes. ( A ) Top: polysome profile of 9.8 OD 254 units of extract obtained from a pellet of ∼30 × 10 6 primary astrocytes using 2% DDM instead of 1% triton X-100 + 1% DOC as a single modification of the ‘standard’ extraction protocol. Bottom: separation of a sample of each of the gradient fractions by 1% agarose-TAE gel electrophoresis, followed by EtBr staining. 18S and 28S rRNA bands are indicated. ( B ) Effect of extract volume and range of sucrose percentage on polysome profiles. The ‘standard’ extraction method was applied on HeLa cells to generate three samples, each containing 10 OD 254 extract units in three different volumes, which were loaded on three different gradients as follows: 1 ml extract on 10.5 ml of 10–50% sucrose gradient (black); 2 ml extract on 9.5 ml of 13.8–50% sucrose gradient (dashed light gray); and 2.5 ml extract on 9 ml of 15.7–50% sucrose gradient (dashed dark gray). ( C ) Top: polysome profiles of ∼10 × 10 6 primary astrocytes performed using ‘standard’ (blue) or ‘optimized’ (black) extraction method. Bottom: as explained in A. The indicated 18S and 28S rRNA bands were used to quantify distribution of ribosomal complexes along the gradient. ( D ) As explained in C, except that ∼10 × 10 6 of HeLa cells were used. ( E ) The gradient fractions presented in (C) (as indicated) were subjected to 12% polyacrylamide-SDS-PAGE followed by immunoblot analysis using antibodies specific for Calnexin, Sec61a, ribosomal proteins S6 (RPS6) and L26 (RPL26).
Article Snippet: For western immunoblot analysis following 12%-acrylamide-SDS-PAGE, the following antibodies were used: Rabbit anti-calnexin (sigma aldrich c4731, 1:5000), mouse anti-sec61a (santa cruz sc-393182, 1:1000), mouse anti-RPS6 (cell signaling #2217, 1:1000), Rabbit anti-RLP26 (abcam ab59567, 1:4000), mouse anti p-S6K (Cell Signalling #9206, 1:1000), rabbit anti t-S6K (Cell Signalling #2708, 1:1000).
Techniques: Extraction, Membrane, Modification, Nucleic Acid Electrophoresis, Staining, SDS Page, Western Blot
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