There are a lot of different methods available for protein purification from various solutions, e.g. cell lysates, body fluids and tissue homogenates. One of the most efficient is immunoaffinity purification using a specific antibody to capture the protein of your interest. This procedure is performed either with immunoaffinity columns or by immunoprecipitation of an antibody/protein/bead complex by centrifugation. As the most efficient, easy-to-use and reliable method, I use magnetic beads from Dynal coated with protein A or G to capture antibody/protein complexes out of different protein-containing homogenates with a reusable magnet. No additional equipment (column/centrifuge) and little hands-on time are required.
1 or 5 ml of beads are provided in a tube with PBS (pH 7.4)/sodium azide so that each user can choose the amount of beads necessary for their procedure. The amount of beads required depends on the affinity of the antibody used to capture the protein of interest (this has to be established empirically with different antibody/antigen concentrations) as well as ability of the capture antibody to bind to the beads. The magnetic beads (2.8 µm diameter) have a high surface area for maximal antibody binding capacity (3-9 m2/109 Dynabeads®); I calculated that to be 7 µg of antibody/ml homogenate for 50 µl of beads.
To optimize, I tested different concentrations of antibodies and beads for protein A/polyclonal antibody and protein G/monoclonal mouse IgG1. (Protein A or G binding recommendations for different antibody species are provided in the manuals). In the end, I used 2x more beads with the monoclonal antibody/protein G beads than the polyclonal/protein A beads, while keeping the same amount of protein lysate (homogenate from rat cortex).
For usage, beads are resuspended by vortexing and the required amount is transferred into a new polypropylene plastic 1.5 ml tube for washing with PBS (neutral pH). To remove the washing solution, tubes were placed on magnet, so that the brown beads move quickly (1-2 min) to the wall on the side of the tube and the washing solution can be removed carefully from the tube. Magnets for 1.5/2 ml, 15 ml or 50 ml tubes are available. Once the beads are removed with the magnet, the appropriate amount of antibody is added to the homogenate and incubated with the solution rotating upside-down in the cold. To isolate membrane protein complexes out of rat cortex homogenate, I increased the volume of the homogenate (because of its high viscosity) before I incubated 250 µl beads with 5 ml homogenate in order to reduce any non-specific binding of proteins to the beads. As the incubation time depends upon the particular antibody/protein system, it has to be established for each system empirically. I extended the recommended incubation from 1 h to 4 h. After adding the protein A or G Dynabeads®, I incubated for an extended time: 8 h or overnight (4°C) due to the viscous solution and the large volume. Since the solution was viscous, removal of the beads to the side of the tube was slow for the first 2 washes of the beads with PBS (30 min instead of the expected 1-2 min), but shortened after clearance of the solution. Elution of the proteins is recommended by using either low pH (0.1% citric acid, pH 2-3), boiling in SDS or solutions with higher ionic strength. As I planned further downstream immunoprecipitations, I eluted with 1% NP40 in PBS (pH 7.4). I used this method with different polyclonal and monoclonal antibodies and had fairly good protein yields from the purified rat brain membranes.
Dynabeads® might be more expensive than other systems, but they are very versatile. It is also possible to permanently couple the antibody to the beads and reuse the same system after elution of the proteins, depending on the user’s applications.