Inexpensive synthetic-based matrix for both conventional and rapid purification of protein A- and tandem affinity purification-tagged proteins

Immunoglobulin G (IgG)–Sepharose is often used for purification of protein A- and tandem affinity purification (TAP)-tagged proteins from eukaryotic cells, but because it is based on an agarose matrix, it is not always optimal for all proteins. Synthetic matrices such as IgG–Dynabeads have improved properties over IgG–Sepharose but are generally expensive. Here we describe the preparation and properties of an IgG matrix based on Fractogel EMD beads. As a synthetic-based matrix, IgG–Fractogel has clear advantages over IgG–Sepharose. IgG–Fractogel can also be used in applications that usually use IgG–Dynabeads but at a significantly reduced cost.

PAGE sample buffer added to resulting supernatant and bead fractions. All samples were boiled for 5 min, run on 8% SDS PAGE, transferred to nitrocellulose membrane and probed with anti-Tea1 antibodies to assess efficiency of TEV cleavage and release of Tea1 from the beads.
We note that although there are differences between the strains and methods of extract preparation in the two specific examples shown in Fig. 1, further experiments indicate that these did not effect the differential release of Tea1 from the two different types of beads.

Preparation of IgG-matrices
IgG-Fractogel was prepared as described in the accompanying protocol. Fractogel without IgG (Fig. 2B) was prepared using the same partial deactivation of reactive groups used in preparation of IgG-Fractogel, and the beads were kept stored in dH2O at 4°C. IgG-Dynabeads were prepared as described by Oeffinger et al. [4].

Single-step purification of TAP-tagged proteins on IgG Fractogel and IgG-Dynabeads
For the experiments shown in Fig. 2A, wild-type cells (strain KS515) and cells expressing Mto1-TAP (C-terminal TAP tag, consisting of RNAse A S-peptide, TEV cleaveage site, and ZZ domain; [3]) from the endogenous mto1+ chromosomal locus (strain KS3524) were grown in 4xYES at 30° C and harvested in exponential phase at OD 595 15. The cell pellet was washed twice in 1/6 culture volume ice-cold 10 mM sodium phosphate pH 7.5, 0.5 mM EDTA, resuspended in a final volume of 0.3 ml of the same buffer per g of wet cell mass and snap frozen as pellets in liquid nitrogen. Cells were ground in liquid nitrogen using the Retsch grinder. The resulting frozen cell powder was resuspended in 2 ml ice cold extract buffer per 1 g of frozen cell powder. Extract buffer was 10 mM sodium phosphate pH 7.5, 100 mM sodium chloride, 0.5 mM magnesium chloride, 1.5 mM EGTA, and 5% glycerol, with protease inhibitors 1 mM PMSF, 1mM AEBSF, 1 mM benzamidine, 5 µg/ml chymostatin, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin, and 5 µg/ml E64. Triton X-100 was added to 1% final (v/v) and the extract was briefly centrifuged twice at 3,200 x g for 5 min to remove unbroken cells and large debris.
Purification methods were modelled on the methods of Oeffinger et al. [4]. To compare behavior of IgG-Dynabeads vs. IgG-Fractogel, 2 ml of extract from either wild-type or Mto1-TAP-expressing cells was mixed with either 1.5 x 10 8 IgG-Dynabeads or 25 (=1X) or 50 (=2x) µl of packed IgG-Fractogel beads and incubated with gentle mixing at 4°C for 45 min. Beads were then washed quickly using either a magnet (Dynabeads) or by gravity flow in a disposable column (Sigma C2353; Fractogel). Washes consisted of 2 x 4 ml extract buffer containing 1% TX-100 (without protease inhibitors), 2 x 4ml extract buffer containing 0.1% TX-100 (without protease inhibitors), and 1 x 4 ml of ammonium acetate pre-elution buffer (0.1 M ammonium acetate, 0.1 mM magnesium chloride, 0.2% Tween-20). Washes used a "jet" of liquid from a Gilson Pipetman P5000, to resuspend beads. Beads were then collected and washed in 1.2 ml of ammonium acetate pre-elution buffer, and eluted twice for 20 min each with 0.5 ml of 0.5 M ammonium hydroxide, 0.5 mM EDTA. The two elutions were combined, lyophilized, resuspended in SDS-PAGE sample buffer and run on 10% SDS-PAGE.
To compare protein-binding to IgG-Fractogel vs. Fractogel without IgG (Fig.  2B), 2 ml of extract from either wild-type or Mto1-TAP strains was mixed with either 25 µl packed beads of IgG-Fractogel or Fractogel without IgG and incubated with gentle mixing at 4°C for 30 min. Beads were then washed and further processed as described immediately above.
Purification of the budding yeast APC (Fig. 2C) was done essentially as described above for fission yeast Mto1, except that strains JB811 (untagged negativecontrol strain) and SJ148.2 Apc4-SZZ tub2-401 (tagged APC strain) were used (both were the kind gift of Dr. Kevin Hardwick, University of Edinburgh). Either 1.5 x 10 8 IgG-Dynabeads or 25 µl of packed IgG-Fractogel beads were used in the pull-downs.