> Last Item Added:
0 Items

Millipore Technical Publications


Protocol: Affinity Purification with Ultrafree-MC Centrifugal Filters
Catalogue Number:
Year:2005
Rev Date:06/01/2005
Introduction

Affinity interaction chromatography is often the single most effective step in any protein purification procedure. Up to 95% purity can be achieved in one step, depending on the nature of the interaction and the starting composition of the protein solution. Well known examples of highly specific affinity interactions include antibodies and protein A/G; multiple histidine tags and nickel; streptavidin and biotin; antibodies and antigens; and many others. Less specific interactions are also used for enrichment or depletion protocols, including albumin depletion on cibacron blue resin, glycoprotein enrichment on concavalin A resin, and capture of nucleic acid-binding proteins on heparin resin.

Affinity chromatography 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 a microfuge tube that contains 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 eluant

Although the method is relatively simple, care must be taken not to remove the chromatography resin when pipetting off the supernatant.

Pre-packed mini-spin columns are a convenient tool for small-scale protein purification. They are operated by centrifugation and usually require less than an hour for the whole procedure.

Another alternative for small-scale purification are centrifugal devices with microporous membrane, such as Ultrafree-MC centrifugal devices. The sample can be added to the filter basket and mixed for the needed residence time and then centrifuged. The process removes the interstitial liquid but does not dehydrate the beads. Washing and elution can also be performed in a similar manner and are more effective due to the efficient removal of buffer and/or eluant.

Ultrafree-MC centrifugal filter units with microporous membrane (Figure 1.1) come with low protein-binding Durapore PVDF membrane in five different pore sizes from 0.1 to 5.0 µm. Affinity resin can be loaded into the filter basket and the device used as a “home-made” mini-spin column.

We show the applicability of the device for purification of rabbit IgG on PROSEP-A resin and His-tagged C-RP protein on three different commercial metal-chelate resins.

Figure 1.1 Ultrafree-MC centrifugal filter unit

Materials
  • Ultrafree-MC 0.45 µm centrifugal devices (Millipore cat. No. UFC3 0HV 00)
  • PROSEP-A high capacity resin (Millipore cat. No. 1131 118 26)
  • Rabbit serum Gibco (Invitrogen lot no. 1132782)
  • Micro-centrifuge Biofuge® Pico (Heraeus instruments)
  • Jouan CR1822 fixed angle rotor centrifuge
  • Xcell SureLock™ Mini-cell vertical electrophoresis system (Invitrogen cat. No. EI0001)
  • NuPage® NOVEX Bis-Tris 4 –12%, 1 mm thick, 15 well SDS gels, (Invitrogen cat. No. NP0323)
  • NuPage Sample Reducing agent (10X) (Invitrogen cat. No. NP009)
  • NuPage SDS Sample Buffer (4X) (Invitrogen cat. No. NP007)
  • SimplyBlue™ SafeStain Coomassie G-250 stain (Invitrogen cat. No. LC6060)

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-A media 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 of 400 µL each were performed by adding 400 µL of binding buffer A and centrifuging at 2,000 x g for 2 minutes each.
  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
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. Recombinant proteins were expressed in Escherichia coli.
  2. Cells were prepared at a 10X concentration using lysis buffer. Lysozyme was added to a concentration of 0.1 mg/mL. To reduce the viscosity, benzonase was added to the lysate. The lysates were clarified by centrifugation.
  3. 200 µL of the 50% resin slurry were added to the Ultrafree-MC device and the residual fluid was removed by centrifugation for 1 minute at 500 x g.
  4. The resin was equilibrated with 500 µL of binding buffer and centrifuged for 2 minutes at 500 x g.
  5. 500 µL of the clarified lysate were added to the resin.
  6. The His-tagged proteins were bound for 30 minutes with light agitation.
  7. The lysate was removed by centrifugation at 500 x g for 10 minutes.
  8. The resin was washed with 500 µL of wash buffer for 5 minutes with agitation. The wash solution was removed by centrifugation for 5 minutes at 500 x g. This step was repeated two more times.
  9. 250 µL of elution buffer were added to the Ultrafree-MC device and mixed for 5 minutes. Purified protein was recovered by centrifugation at 500 x g for 1 minute.
Results

The results of purifying rabbit IgG using Ultrafree-MC centrifugal devices are shown in Figure 1.2. The device was challenged with approximately 14 mg of total protein, with an estimated IgG content of 1. 5–2 mg. The original serum, flow-through, three washes and two eluted fractions were analyzed by SDS-PAGE. The total amount of purified IgG, as estimated by OD280, was 1.2 mg and 1.1 mg on two devices processed in parallel. The whole procedure was completed in less than 1 hour. This method can be useful for monitoring the titer of antigen-specific antibodies after immune activation, or whenever small amounts of IgG need to be purified.

Figure 1.3 shows the results of His-tagged protein purification in Ultrafree-MC devices using Ni-NTA agarose and BD Talon™ resin. Two recombinant proteins were purified: C-RP, highexpressing protein of 26 kDa; and RT66, lowexpressing protein of 66 kDa. As seen in Figure 1.3, both purifications resulted in high purity proteins. The amount of proteins purified out of 500 µL of lysate was 32–35 µg for C-RP protein and 8 µg for RT66.

The data show that affinity batch purification can be effectively performed on a small scale using Ultrafree-MC devices loaded with resin. The process combines 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 of resin to lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. This method is applicable to recombinant protein purification, antibody purification and immunoprecipitation.

Figure 1.2 Rabbit IgG purification on PROSEP-A resin in Ultrafree-MC devices.


Lane 1: Molecular weight standards
Lane 2: Rabbit serum
Lane 3: Flow through
Lanes 4 – 6: Three consecutive washes
Lanes 7, 8: Eluted IgG from two devices

Figure 1.3 His-tagged protein purification using Ultrafree-MC devices.


Lane 1: Molecular weight standards
Lane 2: E. coli lysate expressing C-RP protein
Lane 3: E. coli lysate expressing RT66 protein
Lanes 4, 5: Proteins purified on Ni-NTA resin
Lanes 6, 7: Proteins purified on BD Talon resin