============================= His-tag protein purification ============================= .. warning:: This protocol was attempted for Tn5 purification. This was unsuccessful, though possibly not because of this protocol. Source ------ The `QIAexpressionist `__ is the go-to manual on His-tag purifying proteins. Required solutions ------------------- * **Lysis buffer**: You have options! Check page 113 of the QIAexpressionist. * **10% PEI**: pH to 7.2 .. note:: Do not use the cOmplete running and elution buffers with normal Ni-NTA His-tag resin! They contain chelating agents that will remove the nickel ions! cOmplete His-tag resin is resistant to small concentrations of chelating agents. * **cOmplete running buffer (native)**: ===================== ================ ================== Component Concentration g/L final volume ===================== ================ ================== DI water 90% HEPES 20mM 4.766 g NaCl 800 mM 46.762 g Imidazole 20 mM 1.362 g EDTA 1 mM 0.292 g DTT 2 mM 0.3085 g Glycerol 10% NaOH to pH 7.2 ===================== ================ ================== .. note:: Perform the pH after all components have been added. * **cOmplete elution buffer (native)**: ===================== ================ ================== ================================================================ Component Concentration g/L final volume Purpose ===================== ================ ================== ================================================================ DI water 90% HEPES 20mM 4.766 g Main buffer component NaCl 800 mM 46.762 g High salt maintains protein solubility Imidazole 300 mM 20.42 g Prevents non-specific binding to column. EDTA 1 mM 0.292 g Chelating agent, deactivates proteases and other enzymes. DTT 2 mM 0.3085 g Reducing agent, prevents nonspecific cystine crosslinking. Glycerol 10% Prevents hydrophobic protein-protein interactions. NaOH to pH 7.2 ===================== ================ ================== ================================================================ .. note:: Perform the pH after all components have been added. * **NI-NTA running buffer (native)**: ===================== ================ ================== =================================================================== Component Concentration g/L final volume Purpose ===================== ================ ================== =================================================================== DI water 90% NaH2PO4 50mM 6.90 g Main buffer component. NaCl 800 mM 46.762 g High salt maintains protein solubility. Imidazole 15 mM 1.022 g Prevents non-specific binding to column. beta-mercaptoethanol 5 mM Weaker reducing agent, prevents nonspecific cystine crosslinking. Glycerol 10% Prevents hydrophobic protein-protein interactions. NaOH to pH 8.0 ===================== ================ ================== =================================================================== .. note:: Perform the pH after all components have been added. Protocol -------- 1. Resuspend the cell pellet in lysis buffer, on ice. For every gram of cell pellet, use 10 mL lysis buffer. 2. Sonicate the resuspended cells, using 5 cycles of 30-second, medium-amplitude 50% duty-cycle sonication. 3. Spin to clarify the lysate using maximum centrifuge speed (14600 xg) for 30 minutes. 4. If the protein of interest has DNA-binding activity, add 1.2 mL/10mL **10% PEI** dropwise while stirring. Centrifuge at maximum speed for 30 minutes to remove bound DNA. 5. Fill a column with roughly 1.0 mL resin per gram of cell lysate. .. note:: When using affinity columns, the volumes required will be listed as Column Volumes (CVs). The amount of resin is the CV. For a gram preparation, this would mean the CV is 1 mL. 5. Equilibrate the column with 3 CVs of running buffer. 6. Flow the clarified cell lysate across the column, collecting the flow-through. 7. Rinse the column with 8 CVs of running buffer, collecting flow-through fractions. 8. Elute with four separate 0.5 CV washes of elution buffer. 9. Run all collected samples on a SDS-page gel to confirm successful purification.