Protein purification by affinity chromatography

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Protein purification by affinity chromatography

Procedure

In order to perform protein purification by affinity chromatography, the following steps must be followed:-

Preparing Ni-NTA Column (Equilibriation Step)

NOTE: One can use a pre-packed Ni-NTA column or can pack it manually.

When preparing a column as described below, make sure that the snap-off cap at the bottom of the column remains intact. To prepare a column:

  1. Resuspend the Ni-NTA Agarose in its bottle by inverting and gently tapping the bottle repeatedly.
  2. Pipet or pour 1.5 ml of the resin into a 10-ml Purification Column. Allow the resin to settle completely by gravity (5–10 minutes) or gently pellet it by low-speed centrifugation (1 minute at 800 × g). Gently aspirate the supernatant.
  3. Add 6 ml sterile, distilled water and resuspend the resin by alternately inverting and gently tapping the column.
  4. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant.
  5. Add 6 ml Buffer A (buffer used for the lysis of the cells).
  6. Resuspend the resin by alternately inverting and gently tapping the column.
  7. Allow the resin to settle using gravity or centrifugation as described in Step 2, and gently aspirate the supernatant.
  8. Repeat Steps 5 through 7.

Storing Prepared Columns

To store a column containing resin either manually packed or pre-packed, add 20% ethanol as a preservative and cap or parafilm the column. Storing of column should be at 4°C.

Purification of proteins

Using the native buffers, columns and cell lysate, follow the procedure below to purify proteins under native conditions:

  1. Add 5-10 ml cell lysate in a manually prepared or pre-packed Ni-NTA purification column.
  1. Pass the Buffer A in the column at a speed of 2 ml/min and incubate for 5-10 minutes for proper binding of the proteins.
  1. Settle the resin by gravity or low-speed centrifugation (800 × g), and carefully aspirate the supernatant in case of a manually prepared column. Save supernatant at 4°C for SDS-PAGE analysis.
  2. Load the remaining cell lysate same as mentioned in Step (1-3).
  3. Wash the column with Buffer A (2ml/min) for 30-40 minutes.
  4. Elute the protein of interest by passing the Elution Buffer (Buffer B) in a gradient. Collect 1-2 ml fractions and analyze them with SDS-PAGE.

Note: Store the eluted fractions at 4°C. If -20°C storage is required, add glycerol to the fractions. For long-term storage, add protease inhibitors to the fractions.

If one wishes to reuse the resin or pre-packed column to purify the same or different recombinant protein, wash the resin/column with 1 M NaOH for 30 minutes and equilibrate the resin/column in a suitable binding buffer.