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文献和实验6xHis-tagged protein purification using Qiagen Ni-NTA Column under Native Condit
.0 by NaOHElution Buffer (1 liter)50 mM NaH2PO4 6.90g NaH2PO4.H2O(MW 137.99 )300 mM NaCl17.54g NaCl (MW 58.44 )or 60ml 5 M250 mM Imidazole 17.00g Imidazole (MW 68.08 )Adjust pH to 8.0 by NaOHPreparing cleared lysates under Native Conditions1.Thaw the cell pellet
Blue Native Gel Electrophoresis
% glycerol, 25 mM BisTrsi-HCl, pH7.0 Mix gently Load 5.5 ul Protein marker Mr (kDa) Ferritin 880, 440 β-amylase 200 BSA 132, 66 β-lactoglobulin 35 Thyroglobulin (670kD) migrates at position close to ferritin dimer (880kD).
Determination of target protein solubility
Expression 1. Resuspend cell pellet in 5 ml of lysis buffer for native purification. 2. Freeze sample in dry ice/ethanol, and thaw in cold water. Alternatively, add lysozyme to 1mg/ml and incubate on ice for 30 min. 3. Sonicate
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