Purification of recombinant green fluorescent protein by three-phase partitioning |
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Authors: | Jain Sulakshana Singh Rajni Gupta M N |
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Affiliation: | Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India. |
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Abstract: | The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step. TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts. In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase. When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification. The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band. The fluorescence excitation and emission spectra agreed with values reported in literature. |
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Keywords: | Three-phase partitioning Green fluorescent protein Proteins |
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