Affinity Purification with Buffer Exchange and Concentration: Amicon® Pro System

Traditional centrifugation-based affinity purification presents certain challenges. Batch processing of resins directly in microcentrifuge tubes is subject to sample loss during aspiration. Spin columns alleviate this concern; however, due to their limited volume capacity (typically 0.5 mL), the protocol is tedious, requiring multiple spin cycles during the wash and elution steps. Moreover, while most purification workflows require additional sample neutralization, buffer exchange, and/or concentration prior to downstream analysis, no single traditional centrifugation device is capable of performing all steps in the purification process.

The Amicon® Pro purification system is an adaptable centrifugal device that couples affinity-based spin column purification with downstream sample concentration and buffer exchange. By condensing the protein preparation workflow, the Amicon® Pro system eliminates the need for multiple sample transfers, thereby minimizing protein loss.

Amicon® Pro Protocol for Affinity Purification

This protocol was derived from the study described in the application note, The Amicon® Pro purification system: condensing the protein purification workflow into one centrifugal device. The method was designed for the affinity purification of GST- or His-tagged recombinant protein (from 0.5 mL of lysate).

NOTE: For all protocols using the Amicon® Pro device, all steps, with the exception of binding reactions, are spin-based. A swinging bucket rotor is required for all steps with the exception of the reverse spin.

Add 100 µL Resin (50% Ni-NTA agarose or glutathione agarose slurry) and 1.5 mL Wash buffer to exchange reservoir. Spin 1000 x g for 1 min.

  1. Add 0.5 mL sample and mix with resin by pipetting. Incubate with upright agitation in an orbital shaker for 1 h. [Note: end-over-end mixing may be less effective, because residual resin on the wall and cap may not be collected efficiently.]
  2. Spin 1000 x g for 1 min to clear unbound species.
  3. Add 1.5 mL Wash buffer. Spin 1000 x g for 1 min.
  4. Attach the Amicon® Ultra 0.5 mL filter (10k MWCO).
  5. Add 1 mL Elution Buffer and mix with resin. Incubate for 5 min.
  6. Spin at 4000 x g for 15 min.
  7. Add 1.5 mL desired buffer. Spin 4000 x g for 15 min to exchange buffer and concentrate.
  8. Recover purified protein from the Amicon® Ultra 0.5 filter by reverse spin.

Results

33% faster, less hands-on time. The Amicon® Pro device offered a significant reduction in overall processing time (33%, a savings of 63 min) while also minimizing hands-on time by decreasing the spin steps (3 to 1, in each case) required during the wash, elution, concentration, and buffer exchange phases. SDS-PAGE showed that, despite fewer washes in the Amicon® Pro protocol, high yield and purity were maintained (See figure below).

High yield and protein purity from Amicon® Pro purification compared to two traditional affinity purification schemes

High yield and protein purity from Amicon® Pro purification compared to two traditional affinity purification schemes. In each case, 100 µL settled resin was used to purify His-CRP from 0.5 mL E coli lysate. A representative gel shows the various fractions derived during purification using the three bind-wash-elute methods: Amicon® Pro device, 0.5 mL spin column, and slurry batch purification method using Ni-NTA resin. Detailed methods can be found in the application note.

Elution without dilution. For the elution phase, a single one-minute spin was sufficient to release >90% of bound protein (See figure below). Moreover, when the included Amicon® Ultra 0.5 mL filter was attached, the sample underwent simultaneous concentration during the elution step. The fraction’s final concentrated volume was, on average, 45 µL following a 15 minute spin. By combining these two steps, the Amicon® Pro device further eliminated the tedious requirement of determining protein content across the various eluted fractions before concentrating.

Protein elution without sample dilution

Protein elution without sample dilution. 100 µL Ni-NTA agarose resin and 0.5 mL His-CRP lysate were added to Amicon® Pro or traditional devices (n = 3) and incubated for 1 hour. After clearance and washing, proteins were eluted from spin devices, either once in 1.0 mL Elution Buffer (Amicon® Pro) or 3 times with 500 µL Elution Buffer (Competitor spin column). While three elution steps were required for the smaller spin columns, a single spin in the Amicon® Pro device was sufficient to recover > 90% of affinity-captured protein in an average of 45 µL final volume*.

*For these samples, the included Amicon® Ultra 0.5 mL filters were attached, so that the samples underwent simultaneous concentration during the elution step. The fraction’s final concentrated volume was, on average, 45 µL following a 15 minute spin.