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. 2015 Apr 8;4:e04659. doi: 10.7554/eLife.04659

Figure 1. Purification of tRNA-interacting proteins with immobilized tRNA-resin.

(A) A schematic diagram for preparation of tRNA-agarose by hydrazide coupling. (B) tRNA-binding proteins purified with the tRNA-agarose were analyzed by SDS-PAGE/CBB staining. Purification was performed in the absence (−) or presence (+) of 3 mM Mg-ATP. Bands appearing mainly in the ATP minus or plus lanes are marked by closed and open arrowheads, respectively. Bands that were identified by peptide mass fingerprinting after in-gel digestion are indicated by small numbered arrows. (C) A summary of the proteins identified in B. The numbers correspond to the arrows shown in B. *Sro9p, with a calculated molecular mass of 48.1 kDa, is known to migrate as a 60-kDa band on SDS-PAGE (Sobel and Wolin, 1999). (D) Yeast lysates prepared from strains expressing either Ssa1p-FLAG or Ssa2p-FLAG in addition to the wild type-strain were subjected to immunoprecipitation with anti-FLAG agarose in the absence (−) or presence (+) of 3 mM Mg-ATP. One-tenth of the eluates were analyzed by Western blotting with the anti-FLAG antibody (WB), and the remainders of the eluates were subjected to RNA extraction and Northern blotting with a probe against mature tRNA-ProUGG (NB).

DOI: http://dx.doi.org/10.7554/eLife.04659.003

Figure 1.

Figure 1—figure supplement 1. Identification of Ssa1p/Ssa2p as tRNA-binding protein by peptide mass fingerprinting.

Figure 1—figure supplement 1.

(A) The band marked as ‘1’ in Figure 1B was excised from the gel and in-gel-digested with trypsin. The resulting peptides were recovered by extraction with 30% vol/vol acetonitrile, 0.1% vol/vol TFA, and subjected to MALDI/TOF MS (Voyager DE, Applied Biosystems, Foster City, CA). Peaks with red numbers indicate those derived from Ssa1p/Ssa2p. Protein identification was carried out with MS-Fit software (http://jpsl.ludwig.edu.au/ucsfhtml3.4/msfit.html). (B) MS data were compared with the theoretical molecular masses of tryptic fragments from Ssa1p and Ssa2p. (C) A schematic drawing of the domain structures of Ssa1p and Ssa2p. The amino-acid positions of domain boundaries are indicated. The numbers between the bars indicate the identity of the two amino acid sequences.
DOI: http://dx.doi.org/10.7554/eLife.04659.004