Affinity Purification with Ultrafree^®-MC Centrifugal Filters

Affinity interaction chromatography is an effective method for protein purification that can achieve up to 95% purity in one step. It is often employed in the small-scale batch mode as a quick method for microgram-scale protein purification. The typical protocol involves:

1. Pipetting a small volume of affinity resin into the microfuge tube containing the sample
2. Vortexing the tube for a few minutes
3. Centrifuging the resin to the bottom
4. Pipetting off the supernatant
5. Washing a few times (using steps 2 and 3)
6. Eluting with a small amount of eluent

Pre-packed mini-spin columns for protein purification simplify this process into a one-hour protocol.

For a more flexible protocol, centrifugal devices with a microporous membrane, such as Ultrafree^®-MC centrifugal devices, hold the sample and affinity resin in a filter basket, thus creating a “home-made” mini-spin column. These devices combine the high efficiency of batch binding and washing with the handling convenience of a mini-spin column.

With minimal hands-on time, the method provides flexibility in resin-to-lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. Here, the method was used to purify rabbit IgG on ProSep^® resin and His-tagged C-RP protein on three different commercial metal-chelate resins.

Method for IgG Purification

Solutions

• ProSep-A binding buffer A: 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0
• ProSep-A elution buffer B2: 0.2 M Glycine/HCl, pH 2.5
• ProSep-A neutralization buffer: 1 M Tris/HCl, pH 9.0

Procedure

1. 200 mg of ProSep^® resin were placed in Ultrafree^®-MC 0.45 μm filter basket.
2. The columns were equilibrated with 400 μL of binding buffer A and centrifuged for 1 minute at 100 x g.
3. 200 μL of rabbit serum were diluted 1:1 with binding buffer and the entire volume was loaded into the spin column containing PROSEP-A resin.
4. Devices were placed on a shaker for 15 minutes at room temperature and centrifuged at 100 x g for 5 minutes. Flow-through was collected for future analysis.
5. Three consecutive washes were performed, each time adding 400 μL of binding buffer A and centrifuging at 2,000 x g for 2 minutes.
6. 200 μL of elution buffer B2 were added and centrifuged for 2 minutes at 2,000 x g.
7. 26 μL of neutralization buffer were added to each collection tube. A second elution was collected after repeating the same process one more time.

Method for His-tagged C-RP Purification using Ultrafree^® devices

Solutions

• Lysis buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
• Binding buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 10 mM immidazole, pH 7
• Wash buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 20 mM immidazole, pH 7
• Elution buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 250 mM immidazole, pH 7
• 1 mg/mL Lysozyme stock
• Benzonase

Procedure

1. Express recombinant proteins in Escherichia coli.
2. Prepare at a 10X concentration using lysis buffer. (Pellet 100 mL of culture, resuspend in 10 mL of lysis buffer.)
3. Add lysozyme to a concentration of 0.1 mg/mL and Benzonase^® nuclease to reduce the viscosity of the lysate.
4. Clarify the lysate by centrifugation (10 min at 10,000 x g).
5. Add 200 μL of 50% resin slurry to the Ultrafree^®-MC device. Spin the device for 1 min at 500 x g to remove any residual fluid of the resin.
6. Equilibrate the resin with 500 μL of binding buffer and centrifuge for 2 minutes at 500 x g.
7. Add 500 μL of the clarified lysate to the resin and incubate for 30 min with light agitation.
8. Spin the device at 500 x g for 10 min to remove the unbound lysate proteins.
9. Wash the resin with 500 μL of wash buffer and incubate for 5 minutes with agitation. Spin the device for 5 minutes at 500 x g.
10. Repeat the washing step two more times.
11. Add 250 μL of elution buffer and mix for 5 minutes.
12. Spin the device for 1 min at 500 x g to recover the purified protein