Buffer Exchange and Concentration Using Amicon® Pro Centrifugal Filters for Continuous Diafiltration

Even more efficient desalting with simultaneous macrosolute concentration can be achieved using the Amicon® Pro device, which was specifically engineered for effective buffer exchange (≥ 99%) with a single spin (see figure below). Furthermore, the Amicon® Pro device enables continuous diafiltration. Continuous diafiltration maintains a constant volume throughout the buffer exchange process, by matching the rate at which exchange buffer is introduced to the filtrate flow. Because the volume remains constant, the concentration of retained solute also remains constant, providing a gentler method of buffer exchange.

As shown in another protocol, buffer exchange is thus completed in a single step, instead of three steps as required for traditional diafiltration methods (see data comparing traditional and continuous diafiltration).

Amicon® Pro device for single-spin desalting and buffer exchange

Amicon® Pro device for single-spin desalting and buffer exchange. Specific design components include: (1) large upper reservoir of the exchange device, (2) tight contour matching of the exchange device to the Amicon® Ultra 0.5 mL filter to minimize internal spacing when the two devices are connected, and (3) tapered exchange device tip for optimal buffering.
 

Amicon® Pro Method for Desalting and Concentration

  1. Select the Amicon® Pro device with the appropriate nominal molecular weight limit (NMWL) for your application.
  2. Optional: Pre-wet device using the particular buffer solution you are using, for example, 0.5 mL TBS-T. Spin 1000 x g for 1 min. In cases of highly dilute samples, a passivation protocol with detergent may be beneficial. In cases of mass spectrometry applications, use mass spectrometry-compatible detergent (such as deoxycholate).
  3. Optional Wash: Add 1.5 mL appropriate buffer. Spin 1000 x g for 1 min.
  4. Attach the included Amicon® Ultra 0.5 mL filter (five MWCO options available) to the exchange device.
  5. Add 1.5 mL* of sample to the exchange device Spin 4000 x g for 15 min to concentrate.
  6. Add 1.5 mL* of desired buffer. Spin 4000 x g for 15 min to exchange buffer and concentrate.
  7. Recover purified protein from the Amicon® Ultra 0.5 filter by reverse spin.

*For larger sample/buffer volumes, spin time will need to be increased appropriately. Please consult user guide for specific instructions.

NOTE: Do not load more than 2 mg total protein in step 5. Exceeding 2 mg will result in protein precipitation upon concentration.
 

Results

With continuous diafiltration, as occurs in the Amicon® Pro device, the sample (after it has undergone initial concentration and has entered the Amicon® Ultra-0.5 mL component of the Amicon® Pro system) remains at a constant volume (and concentration) until the entire quantity loaded in the upper reservoir has been passed through the exchange device tip. In this experiment, over 99.9% of the salt was removed after a single spin (See table below). The Amicon® Pro device also afforded extremely high protein recovery, having, on average, < 2% loss of starting BSA.

  BSA 0.05 mg/mL
Device Amicon® Pro device (with Amicon® Ultra 10 kDa NMWL filter attached)
Spin % Protein Recovery % NaCl Removal
1 Not determined (initial protein concentration step) 0% (NaCl concentration unchanged during proteinconcentration step)
2 >98% >99.9%

Table: Removal of sodium chloride and recovery of protein with Amicon® Pro device.

500 µL of a BSA solution was subjected to simultaneous desalting (using 1 M Tris, pH 7.5) and concentration. The resulting sample was brought to an equivalent volume in 1 M Tris pH 7.5, diluted 1:50 in Milli-Q® H20, and assayed for conductivity (µS/cm) using a standard conductivity meter (InoLab® Cond). Protein concentration was determined using the Direct Detect® Infrared Spectrometer for Protein Quantitation.