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Fig. 3 | SpringerPlus

Fig. 3

From: Preparation of a functional fluorescent human Fas ligand extracellular domain derivative using a three-dimensional structure guided site-specific fluorochrome conjugation

Fig. 3

Purification using high-performance size-exclusion chromatography. a Elution profiles of the partially purified samples and a mixture of them. Top panel FL-5Mal conjugated NFG1CG4-hFasLECD; Middle panel hFasRECD-Fc; Bottom panel the mixture. Used column, Superdex 200 10/300 GL. Elution buffer, 50 mM Tris-hydrochloride plus 150 mM sodium chloride (pH 7.5). Absorbance at 280 nm (blue) and 495 nm (red) was used for the detection. Flow rate, 0.5 ml/min. The retention time of each peak position is shown. The regions shown in underbars were collected and used for the analysis in b. b SDS-PAGE analysis of the purified samples. Lanes: M molecular-weight size markers (14.4, 20.1, 30.0, 42.4, 66.3 and 97.4 kDa), 1–5 purified samples (1, NFG5-hFasLECD; 2 and 3 peak 2 and peak 1 of the FL-5Mal conjugated NFG1CG4-hFasLECD sample, respectively, 4 main peak of the hFasRECD-Fc sample, 5 main peak of the mixture sample), 6–11 receptor-mediated co-immunoprecipitation (6 and 8 purified proteins, 10 buffer only, 7, 9 and 11 co-immunoprecipitated materials; 6 and 7 peak 2 of the FL-5Mal conjugated NFG1CG4-hFasLECD sample, 8 and 9 peak 1 of the FL-5Mal conjugated NFG1CG4-hFasLECD sample, 10 and 11 the buffer only sample). Thick and thin arrows indicate the positions of FL-5Mal conjugated hFasLECD and hFasRECD-Fc, respectively

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