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. Author manuscript; available in PMC: 2015 Apr 25.
Published in final edited form as: J Chromatogr A. 2014 Mar 3;1339:73–79. doi: 10.1016/j.chroma.2014.02.074

Figure 4. Affinity chromatography of GFP-C/EBP.

Figure 4

50 μl of 0.31 mg/ml purified GFP-C/EBP was mixed with 150 μl TE0.1 and applied to a 50 μl rEP18 column prepared with 200 nM rEP18. All subsequent washes were 0.2 ml of the various buffers collected on a microtiter plate. The column was washed 10 times with TE0.1, and then 3 times each with TE0.5, TE1.0, TE1.5, and TE2.0. A, the fluorescence of each fraction was recorded in epifluorescence mode on a Tecan Infinite M200 fluorescence plate reader with λex = 398 nm, λem = 512 nm. For the TE0.5–TE2.0 fractions, the fluorescence of the 3 fractions was summed. B, The fractions shown were concentrated to 20 μl and added to a gel shift assay using radiolabeled EP18. C, A different column containing 1 μM rEP18 was prepared, loaded and eluted as in panel A. L, Loaded sample; FT, unretained fraction; W1–W10, wash fractions; TE0.5–TE2.0, eluates.