Instructions:


1. Column packing


1.1 The temperature of all materials that need to be used should be the same as the temperature of the chromatographic operation, and the liquid is best to be degassed.


1.2 Add distilled water to the lower end of the column to remove the air in the column, close the outlet of the column, and leave a small amount of distilled water in the column.


1.3 When continuously pouring the agarose gel into the column, use a glass rod close to the inner wall of the column for drainage to reduce the generation of bubbles and allow the filler to settle naturally.


1.4 The column pressure does not exceed 0.3 MPa. If the column pressure cannot be measured in the column packing system, the flow rate should be higher than 300 cm/h, but generally only 75% of the maximum flow rate is used in use.


1.5 The packed Ni-Sepharose FF column is equilibrated with 2 ~ 5 column volumes of the initial buffer solution. The recommended flow rate is 100 cm/h. The equilibrated column can be used for loading and washing His-Tag recombinant protein. Depurification.


2. Sample loading


2.1 The sample is generally dissolved in the initial buffer with pH 6 ~ 8. Increasing the pH of the loading buffer can increase the load.


2.2 The buffer should not contain EDTA and citrate, and it is better not to contain reducing agents such as mercaptoethanol and DTT.


2.3 Commonly used buffers include 10 ~ 100 mM sodium phosphate buffer, 20 ~ 200 mM Tris-HCl buffer and so on.


2.4 Generally, 0.15 ~ 0.5 M NaCl should be added to the buffer to eliminate ion exchange.


2.5 When using Nickel-Sepharose FF for the first time, it is recommended to use 50 mM PBS (50 mM NaH2PO4, 0.5 M NaCl, NaOH to adjust pH 7.4) as the initial buffer.


3. Elution


3.1 The most commonly used elution method for nickel-agarose gel FF is to increase the concentration of imidazole for elution.


3.2 Imidazole is alkaline, and pH should be adjusted with HCl after preparation of the corresponding buffer.


3.3 When using Nickel-Sepharose FF for the first time, I don¡¯t know the concentration of eluted imidazole. It is recommended to add 10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM imidazole to the initial buffer. To high, eluted and collected separately, identified by SDS-PAGE electrophoresis and other methods.


3.4 If conditions permit, linear imidazole gradient elution can also be performed to determine the best elution conditions.






Expand applications:


1. Change different chelating ions


1.1 IDA chelated nickel-agarose gel FF can use EDTA to remove the nickel. After removing the nickel, you can clean the gel with 0.1 ~ 1.0M NaOH, and then re-chelate the nickel to make the gel look new. It can also chelate other metal ions, such as Cu2+, Zn2+, Co2+, Fe3+, etc., for various purifications.


1.2 Denickel method: rinse the column with 5-10 column volumes of distilled water, then rinse the column with 5-10 column volumes of 100 mM EDTA, and then use 2-3 column volumes of 0.5 M NaCl to wash away the remaining EDTA .


1.3 Method of chelating metal ions: fully equilibrate the denickel column with 2 ~ 5 column volumes of distilled water, and pass the column with 0.1 ~ 0.3M metal salt solution for 5 ~ 10 column volumes to chelate the metal, and use 5 ~ 10 after chelation Rinse with a column volume of distilled water to remove unchelated metal ions.


1.4 The metal salt can be sulfate or hydrochloride, such as NiSO4, CuSO4, ZnSO4, CoCl2, PbSO4, etc. The metal salt solution should be neutral or weakly acidic, and must be filtered to prevent the metal salt from precipitating on the glue.


1.5 If it is Fe3+, it must be chelated at a low pH (pH 3) to prevent precipitation of Fe3+. Generally, a small amount of hydrochloric acid and sulfuric acid can be added to maintain the pH.


2. Multiple elution methods


2.1 Lower pH elution: Most proteins will be eluted at pH 4 ~ 6, or pH 3 ~ 4, the buffer can be sodium acetate, citric acid or phosphate buffer system.


2.2 Competitive elution: increase linearly or increase the affinity of metal ions in one step, such as 0 ~ 0.5 M imidazole, 0 ~ 50 mM histidine, 0 ~ 2 M NH4Cl. Gradient elution is best performed at a constant pH of the starting buffer.


2.3 Chelating agent elution: Chelating agents such as EDTA and EGTA will interact with metal ions and cause the protein to be eluted. This method cannot separate different proteins. In addition, it will affect protein adsorption and cause the fusion protein to fail to hang on the column.


2.4 In all the above cases, 0.15 ~ 0.5 M NaCl must be added to the buffer to eliminate ion exchange.


2.5 When the chelating ion ligand is Cu2+, there are the following three operation modes.


Lower pH elution:


Loading buffer: 50 mM NaH2PO4, 0.5 M NaCl, pH 7.4


Elution buffer: 50 mM NaH2PO4, 0.5 M NaCl, pH 3.5


Competitive elution:


Loading buffer: 50 mM NaH2PO4, 1 M NaCl, pH 7.4


Elution buffer: 50 mM NaH2PO4, 1 M NH4Cl, pH 7.4


Chelating agent elution:


Loading buffer: 50 mM NaH2PO4, 0.5 M NaCl, pH 7.4


Elution buffer: 50 mM NaH2PO4, 0.5 M NaCl, 50 mM EDTA, pH 7.4


Note: The prepared buffer needs to be adjusted to the required pH with NaOH or HCl. The use of lowering pH and chelating agent elution will cause the metal ions to fall off, and the metal ions will be chelated again next time. The most commonly used is competitive elution.






Regeneration and cleaning:


1. Regeneration of gel


1.1 After multiple uses or the need to replace the chelated metal ions, the glue must be de-nickeled and regenerated. Denickel method: rinse the column with 5-10 column volumes of distilled water, then rinse the column with 5-10 column volumes of 100 mM EDTA, and then use 2-3 column volumes of 0.5 M NaCl to wash away the remaining EDTA.


1.2 The columns that have been used multiple times generally need to be cleaned after denickeling. Cleaning method: reverse cleaning the column with 0.1 ~ 1.0 M NaOH at a flow rate of 50 cm/h for 1 ~ 2 h, which can remove strong binding impurities and also remove the heat source.


1.3 After cleaning, it is necessary to chelate metal ions again. Chelation method: fully equilibrate the denickel column with 2 ~ 5 column volumes of distilled water, and pass the column with 0.1 ~ 0.3M metal salt solution for 5 ~ 10 column volumes to chelate the metal, and use 5 ~ 10 column volumes after chelation Rinse with distilled water to remove unchelated metal ions.


2. Cleaning in place


2.1 To remove the protein adsorbed by ion exchange, wash the column reversely with 2 to 3 columns of 2 M NaCl solution.


2.2 To remove strong hydrophobic proteins and lipids, wash the column with 4 column volumes of 70% ethanol or 30% isopropanol.


2.3 Remove protein precipitation and hydrophobic protein, refer to the regeneration of the gel.






Precautions:

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