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:
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.
Figure 1. shows the SDS-PAGE analysis for rabbit IgG purified using an Ultrafree®-MC centrifugal device. Starting material contained approximately 14 mg of total protein, with an estimated 1.5–2 mg IgG content. The total amount of purified IgG was 1.2 mg and 1.1 mg (estimated by OD280) on two devices processed in parallel. This method can be useful for monitoring the titer of antigen-specific antibodies after immune activation, or whenever small amount of IgG is needed.
Figure 2. shows the SDS-PAGE analysis of His-tagged protein purified using an Ultrafree®-MC devices, with Ni-NTA agarose and BD Talon™ resin. From 500 μL of lysate, two recombinant proteins were purified, 32-35µg of CRP (a 26 kDa, high-expressing protein) and 8 μg of RT66 (a 66 kDa, low-expressing protein). This method is applicable to recombinant protein purification, antibody purification, and immunoprecipitation.
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