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. 2021 Oct 8;10:e70236. doi: 10.7554/eLife.70236

Figure 2. Incorporation of Acd into MBP.

Figure 2.

(A, B) In-gel fluorescence for samples of (A) MBP-295Acd and (B) MBP-322Acd at various points during the purification process. The first lane sample is Precision Plus Dual Stain molecular weight standard. Lanes marked input represent cleared cell lysate. Lanes marked unbound represent the solution removed from the anti-FLAG affinity beads after the binding step. Lanes marked bound represent the sample eluted by FLAG peptide. The exclusion and inclusion of the amino acyl tRNA synthetase in the cell transfection is indicated by – and +, respectively. Excitation by a UV transilluminator at 354 nm was used in conjunction with a fluorescein emission filter. (C, D) The same gels as shown in (A, B) after staining with Coomassie blue. (E, F) Western blots using the same samples as (A, B) but run on a different gel. Lanes are the same as (A, B) except for the first lane in which the MagicMark XP standard was used. (G, H) Fluorescence size-exclusion chromatography of purified protein on a Superdex 200 Increase 5/150 GL column. Acd was excited at 385 nm with fluorescence recorded at 450 nm (G) and tryptophan absorption was measured at 280 nm (H). MBP eluted at 2.1 mL, and the FLAG peptide eluted at 2.5 mL.

Figure 2—source data 1. In Figure 2A, lane 2 is the same as lane 1 shown in the main text figure.
Black arrow indicates the size of full-length MBP-295Acd.
Figure 2—source data 2. In Figure 2B, lane 2 is the same as lane 1 shown in the main text figure.
Black arrow indicates the size of full-length MBP-322Acd.
Figure 2—source data 3. In Figure 2C, lane 2 is the same as lane 1 shown in the main text figure.
Black arrow indicates the size of full-length MBP-295Acd, and red arrow indicates the size of the truncation product.
Figure 2—source data 4. In Figure 2D, lane 2 is the same as lane 1 shown in the main text figure.
Black arrow indicates the size of full-length MBP-322Acd, and red arrow indicates the size of the truncation product.
Figure 2—source data 5. In Figure 2E, lane 2 is the same as lane 1 shown in the main text figure.
Black arrow indicates the size of full-length MBP-295Acd, and red arrow indicates the size of the truncation product.
Figure 2—source data 6. In Figure 2F, lane 2 is the same as lane 1 shown in the main text figure.
Black arrow indicates the size of full-length MBP-322Acd, and red arrow indicates the size of the truncation product.