Nickel Column Protein Purification Protocol

Nickel Column Protein Purification Protocol

Nickel affinity column chromatography, also known as immobilized metal affinity chromatography (IMAC), is a common method for purifying recombinant proteins that are engineered to have a hexahistidine tag (6xHis tag) at one end.

Here’s a general protocol:

Materials and Reagents:

1. Cell culture expressing your His-tagged protein of interest

2. Lysis buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0

3. Wash buffer: 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0

4. Elution buffer: 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0

5. Ni-NTA agarose or similar nickel-affinity resin


1. Collect cells from culture and lyse them in the lysis buffer to release the proteins.

2. Purify the cell lysate by centrifugation.

3. Add Ni-NTA agarose to the clarifed cell lysate and incubate at 4°C for 1-2 hours to allow the binding of His-tagged proteins to Ni-NTA resin.

4. Transfer the resin-lysate mixture to column and let the lysate flow through.

5. Wash the column with wash buffer to remove unbound proteins.

6. Elute the bound proteins with elution buffer.

Please note that this is a general protocol and the specifics can vary depending upon the characteristics of your protein of interest, such as stability, tendency to aggregate, etc. Always carefully design and optimize the purification method to suit your protein of interest. Also, be sure to follow any specific guidelines provided by the manufacturer on the use and care of the nickel-affinity resin.

Dr AF Saeed

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