Purification of recombinant green fluorescent protein by three-phase partitioning.
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.
Jain
Sulakshana
S
Chemistry Department, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110016, India.
Singh
Rajni
R
Gupta
M N
MN
D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins