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文献和实验HIS-tagged protein purification
John Mundy,Institute of Molecular Biology,Copenhagen,Denmark 1 liter cell prep 1)grow 20ml cells O/N37℃ dilute 50X into prewarmed LB,grow to 0.6 OD or about 1hr.Induce w/ 2mM IPTG (238mg/0.5l),grow 3hr,spin 6k GS3 10',freeze at 70℃ 2)resuspend
6xHis-tagged protein purification using Qiagen Ni-NTA Column
)Centrifuge lysate at 10,000xg for 20’ at 40℃to pellet the cellular debris and save supernatant.Add 5μl 2xSB to 5μl Supernatant and store at 200℃for SDS-PAGE analysisBatch purification under Native ConditionsAdd 2.5 ml of the 50% Ni-NTA slurry to 10 ml cleared
Procedures for the Analysis and Purification of His-Tagged Proteins
of the protein of interest with a naturally occurring protein (glutathione S -transferase, maltose binding protein, or protein A) and using their natural affinity to substrates (glutathione, amylose, or immunoglobulins) coupled to columns in the purification step
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