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. 2020 May 20;10(10):e3616. doi: 10.21769/BioProtoc.3616

Figure 3. Immunoblot analysis following the purification of geranylgeranyl reductase enzyme tagged with 3x FLAG-tag (FLAG-ChlP).

Figure 3.

The purification of FLAG-ChlP from Synechocystis was performed according to this protocol. Protein suspension that was loaded in Step D4 (L; solubilized thylakoid membranes of 0.5 µg chlorophyll), the first flowthrough in Step D4 (FT; equal volume to L), the first wash in Step D5 (W1; 1/25 volume of the wash fraction), the second wash in step 6 (W2; 1/25 volume of the wash fraction), and the eluate gained in Step D15 (E; 1/250 volume of total, non-concentrated eluate) were analyzed by SDS gel electrophoresis (A) and by a subsequent immunoblot (B). Prior to Western blotting on a PVDF membrane the gel was stained with SYPRO Orange (Sigma) to detect total proteins (A). Proteins blotted on the membrane were probed with the α-FLAG antibody (Merck) (A); the protein band, representing the isolated FLAG-ChlP, is indicated.