Purification of 6xHis epitope tagged proteins by Ni-NTA-Agarose His标签蛋白纯化
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Low (“Low”)Imidazole Buffer 0.5L
100mM Imidizole 3.4g
5% glycerol 25ml 100% glycerol
50mM Tris-HCl (pH 7.9)50ml 0.5M Tris-HCl (pH 7.9)
0.1% Tween-20 0.5M 100% Tween-20
500mM NaCl50ml 5M NaCl
dH2O Fill to 0.5L (start w/ 350ml)
High (“High”)Imidazole Buffer 0.5L
500mM Imidizole 17.0g
5% glycerol 25ml 100% glycerol
50mM Tris-HCl (pH 7.9)50ml 0.5M Tris-HCl (pH 7.9)
0.1% Tween-20 0.5M 100% Tween-20
500mM NaCl50ml 5M NaCl
dH2O Fill to 0.5L (start w/ 350ml)
Sonication and Solubility Test:
Resuspend 1L worth of bacterial pellet in 30ml Low Buffer
Take 30μl pre-sonication sample
Sonicate 3 pulses at 80% power with 7th floor sonicator,2 min on ice between pulses (NOTE: do not let sonicator tip touch side of tube to reduce frothing)
Take 30μl crude sonicated sample
Dispense into 1.5 ml eppendorf tubes,spin 14k rpm,4℃,30 min
Combine soluble fractions into 1 tube
Take 30μl soluble fraction as post-sonication soluble sample
Test solubility of target protein by SDS-PAGE/Coomassie Blue Stain
Add 10μl 4x Sample Buffer and 2μl ?-Mercaptoethanol to each 30μl sample
Add an equal volume of 4x Sample Buffer to one insoluble pellet,add β-Mercaptoethanol to a final concentration of 5%.
Load 14μl (equals 10μl of fraction)on an appropriate concentration SDS-PAGE gel,electrophorese to separate bands well
Stain with Coomassie Blue for 30 min @ RT with rocking,destain with shreds of brown paper towel to aid in removal of dye from gel
