I recently had the occasion to purify the IgG fraction from a small volume of rabbit serum in order to generate a more concentrated solution of antibody for a specific application. After looking around, I chose the NAb Protein A Spin Chromatography Kit from Pierce. NAb Spin Chromatography Kits contain agarose-coupled antibody-binding proteins (A, G, or L), binding buffer, elution buffer, empty spin columns, and collection tubes. Although not included, 1 M phosphate buffer is recommended to neutralize the eluted IgG.
The protocol is very simple. Coupled agarose is pipetted into the spin column and then washed twice with binding buffer. The IgG-containing sample is then added and allowed to bind for 30 min with agitation. Non-binding proteins are spun through and the agarose washed 3 times. Bound IgG is eluted with 4 x 5-min incubations with elution buffer. Each eluate is then neutralized with a tenth volume phosphate buffer.
Since I had never used these spin columns before, I opted for a couple of practice runs using normal rabbit serum prior to using them on the costly anti-serum. From 2 separate purifications performed on 2 different days, I recovered 495 and 488 ug IgG per 100 ul serum. A Coomassie-stained gel of the eluate indicated a very pure preparation of IgG. Only a trace of serum albumin was visible in addition to the heavy and light IgG chains. Although Pierce recommends 4 separate elutions, I found that about 98% of the bound IgG is eluted with the first 2 elutions.
Overall, the Protein A Kit performed very well and gave good recovery of IgG from rabbit serum. However, there are two issues that deserve some comment.
The first is the price. Granted the kit does exactly what is expected of it and saves a good amount of time, however, each purification costs from $18-26, depending on whether protein A, G, or L is used. This could run into quite an expense if many purifications are required.
The second issue is that each elution is carried out using 0.4 ml elution buffer. This results in a fairly dilute IgG prep once the elutions are combined. Although in my hands, the IgG concentration in the first eluted fraction was about 1 mg/ml, the second elution contained only around 50 ug/ml. After pooling these 2 eluates, the IgG concentration was about 0.5 mg/ml. While this may be sufficiently concentrated for some applications, others may require further reduction in volume. I have found that a spin concentration device of MWCO 100,000 designed for optimal protein recovery works very well and can also be used for buffer exchange. Plan on taking an additional hit in protein recovery, however.
Overall, I would highly recommend the NAb Protein A Spin Chromatography Kit from Pierce for occasional IgG purification from serum.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center