In our hybridoma development lab, we used to purchase bulk Protein A resin and purify samples one at a time. But recently, we have been using the Protein A spin columns from Vivascience and found them to be an enormous advantage in our antibody development process. This is primarily because using the Protein A spin columns it is now possible to purify several possible antibody candidates at the same time. Once purified, the samples are concentrated, run on a zonal gel electrophoresis as well as tested by ELISA against the target antigen to see if they meet the specific criteria. This allows us to quickly choose which candidates will be chosen for scale up and subcloning while any other candidates are discarded, saving valuable time in the process.
Vivascience’s Protein A spin columns are based on the 42 kDa Protein A attached to regenerated cellulose, which is coupled to a proprietary membrane technology located in the polypropylene spin column. Protein A is a component of the cell wall of the bacterium Staphylococcus aureus and specifically binds to the Fc-region of various immunoglobulins (Ig). This makes it a very good candidate to purify a wide range of host antibodies of human, mouse and rabbit origin. However, some subgroups like human IgG3 and mouse IgG1 do not bind to Protein A unless the equilibration and binding conditions are adjusted. The instruction booklet enclosed with the kit outlines these parameters.
To purify a sample, the entire process can be achieved in less than 30 minutes with capacities ranging between 0.2-0.3 mg. I used an Eppendorf mini spin-plus micro-centrifuge to spin the tubes. Any micro-centrifuge that can accommodate 2 mL centrifuge tubes and is capable of speeds between 400 - 800 x g will work. Before you begin the purification process, you first need to know or have a good idea of the approximate IgG concentration in your sample(s) so you do not overload the columns. If the IgG is unknown, you can determine the amount of protein in your sample by running a BCA total protein assay. Second, you have to dilute your sample 1:1 with the appropriate binding buffer that you will use for the purification protocol. This step properly equilibrates the sample to the protein A resin. Once diluted, the maximum volume that can be added to the spin tube is 400 uL, but make sure that your protein load does not over exceed the 300ug capacity of the column.
There are two binding buffers that are recommended for a particular application. A 0.5M NaCl, 10mM K-phosphate, pH 7.4 is used for antibodies that have a high affinity for Protein A and a 3.0M NaCl, 1.5M glycine/HCL, pH 9.0 is used for antibodies that have a low affinity for Protein A. You can refer to the instruction pamphlet enclosed with the spin columns for a list of high and low affinity antibodies to Protein A. The sample that I worked with had a low IgG concentration of ~ 0.5 mg/mL and had a high affinity for Protein A. I diluted my 250uL sample size into 250uL of 0.5M NaCl, 10mM K-phosphate, pH 7.4. I did not filter the sample as it was relatively clear but if necessary, samples can be filtered before protein loading through a 0.45m filter.
To begin the protocol the membrane(s) were first equilibrated by adding 400uL of the appropriate binding buffer to each spin column and the tops were closed. Collection tubes that were sent with the kit were attached to the bottom of the spin tubes and then centrifuged for 5 minutes at 800 x g. Once equilibrated, the diluted sample(s) were loaded onto the membrane, the tops were secured and the previous collection tubes were attached again. The tubes were spun for 7 minutes at 400 x g to allow maximum protein binding to the membrane. After spinning, the collection tubes were removed from the bottom and the flow through was saved as this can be tested later to determine that complete binding was established. Before the elution step is employed, the membranes need to be washed in order to remove unbound contaminants. This was done by adding 400uL of binding buffer onto the membranes, attaching the collection tubes and centrifuging the spin column for 5 minutes at 800 x g. After the wash step is complete, the bound antibodies need to be eluted from the membrane. This is achieved by adding 400uL of elution buffer onto the membrane, adding new collection tubes, and spinning for 5 minutes at 800 x g. After the elution step, 20uL of 1.0M Tris buffer, pH 9.0 was added to the elution fractions to prevent precipitation and maintain antibody activity. Once neutralized, samples were dialyzed against 0.1M phosphate buffered saline, pH 7.4 and concentrated for screening purposes. After dialysis, antibody fractions were read by OD280nm to determine antibody quantity. Yields from the spin columns were between 85-90%.
The price of the spin columns are approximately $100.00 for 24 spin columns or ~ $4.00 per column. If you broke it down on a per mg basis, you are paying ~ $13.40 per mg of protein A bound onto the membrane. Compared to bulk Protein A resin which goes for about $850 for 100 mg, or $8.50 per mg, the price tag on the spin columns is very reasonable. Financially it works great when you are testing 4-5 different monoclonal antibodies and need to get purification results immediately. However, if there is more then 4-5 mg of antibody to purify, it would be much more efficient to purchase Protein A in bulk instead.
One downside of the spin columns is you are bound by a 200-300ug maximum loading capacity onto each membrane. If your specific application calls for more then the 300 ug capacity, then you are forced to run more then one spin column at a time or take the extra time and run them back to back.
Overall, we were very pleased with the simplicity and reproducible results that Vivascience’s Protein A Spin columns offered. You can easily purify several unknown samples with one spin of the centrifuge while saving time and valuable reagents along the way. These are a great addition to our laboratory and I would recommend this product to others.
Thomas Sewell
R&D Applications Specialist
Maine Biotechnology Services