Purification of 6xHis epitope tagged proteins by Ni-NTA-Agarose His标签蛋白纯化

Sugden lab,McArdle Laboratory for Cancer Research, University of Wisconsin-Madison Medical School 

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 NaCl 50ml 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 NaCl 50ml 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 30ul 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 30ul crude sonicated sample

Dispense into 1.5 ml eppendorf tubes, spin 14k rpm, 4oC, 30 min

Combine soluble fractions into 1 tube

Take 30ul soluble fraction as post-sonication soluble sample

Test solubility of target protein by SDS-PAGE/Coomassie Blue Stain

Add 10ul 4x Sample Buffer and 2ul ?-Mercaptoethanol to each 30ul sample

Add an equal volume of 4x Sample Buffer to one insoluble pellet, add
?-Mercaptoethanol to a final concentration of 5%.

Load 14ul (equals 10ul 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