Protein Sample Concentration

Goal

Use the Amicon® Ultra-4 centrifugal filter device concentrate a protein sample through rapid ultrafiltration. The following information and protocol was adapted from here

Before You Start

Picking The Right Filtering Device

The membranes used in Amicon® Ultra devices are characterized by a molecular weight cutoff (MWCO); that is, their ability to retain molecules above a specified molecular weight. Solutes with molecular weights close to the MWCO may be only partially retained. Membrane retention depends on the solute’s molecular size and shape. For most applications, molecular weight is a convenient parameter to use in assessing retention characteristics. For best results, use a membrane with a MWCO at least two times smaller than the molecular weight of the protein solute that one intends to concentrate. See table below (Amicon® User Guide Table 4):

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What You Will Need

• Sample to concentrate
• Swinging-bucket centrifuge (Bio superlab) or fixed-angle rotor with wells/carriers that can accommodate 17 mm × 124 mm 15 mL (location?)
• Conical-bottomed tubes
• P200 or P1000 Pipettor with appropriate tips
• Appropriate MWCO Amicon® Ultra filter device (Amicon® User Guide Figure 1)

CAUTION: To avoid damage to the device during centrifugation, check clearance before spinning.

Tips

Maximize Sample Recovery

Low sample recovery in the concentrate may be due to adsorptive losses, over-concentration, or passage of sample through the membrane.

1. Adsorptive losses depend upon solute concentration, its hydrophobic nature, temperature and time of contact with filter device surfaces, sample composition, and pH. To minimize losses, remove concentrated samples immediately after centrifugal spin.
2. Over-concentration can lead to precipitation and potential sample loss. If the starting sample concentration is high, monitor the centrifugation process in order to avoid over-concentration of the sample.'
3. If the sample appears to be passing through the membrane, choose a lower MWCO Amicon® Ultra-4 device.

Desalting or Diafiltration

Desalting, buffer exchange, or diafiltration are important methods for removing salts or solvents in solutions containing biomolecules.

They can be accomplished by concentrating the sample, then reconstituting the concentrate to the original sample volume with any desired solvent. The process of “washing out” can be repeated until the concentration of the contaminating microsolute has been sufficiently reduced. See process overview below (Amicon® user guide Figure 2).

Procedure

1. Rinse Amicon® Ultra filter device with buffer or Milli-Q® water before use
   • if interference continues, rinse with 0.1 N NaOH followed by a second spin of buffer or Milli-Q® water.
   • CAUTION: Do not allow the membrane in Amicon® Ultra filter devices to dry out once wet. If you are not using the device immediately after rinsing, leave fluid on the membrane until the device is used.
2. Add up to 4 mL of sample (3.5 mL if using a 23° fixed-angle rotor) to the Amicon® Ultra filter device.
3. Place capped filter device into centrifuge rotor
4. Centrifuge setting options:
   1. Swinging-bucket (preferred); counterbalance with a similar device. Spin at 4,000 × g maximum for approximately 10–40 minutes.
   2. Fixed-angle rotor: orient the device with the membrane panel facing up.
      • For Amicon® Ultra 3K, 10K, 30K, and 50K devices, spin at 7,500 × g maximum for approximately 10–40 minutes.
      • For Amicon® Ultra 100K devices, spin at 5,000 × g maximum for approximately 10–20 minutes.

   NOTE: Refer to tables below for typical spin times (Amicon® user guide Table 2 and 3).

5. To recover the concentrated solute, insert a pipettor into the bottom of the filter device and withdraw the sample using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube.

   NOTE: For optimal recovery, remove concentrated sample immediately after centrifugation.