Improved methodology for the affinity isolation of human protein complexes expressed at near endogenous levels

Abstract

An efficient and reliable procedure for the capture of affinity-tagged proteins and associated complexes from human cell lines is reported. Through multiple optimizations, high yield and low background affinity-purifications are achieved from modest quantities of human cells expressing endogenous-level tagged proteins. Isolations of triple-FLAG and GFP-tagged fusion proteins involved in RNA metabolism are presented.

Figure 1. Magnetic beads coupling optimization

Magnetic beads coupled to anti-FLAG M2 antibodies (nos. F3165 and F1804, respectively; Sigma-Aldrich) in three different concentrations—5, 10, and 15 μg/mg of MBs—were used for immunoprecipitation of the reporter protein, BAP-FLAG, in the presence of HEK cell lysate. Anti-FLAG M2 antibodies were either directly coupled to MBs (F3165) or subjected to desalting before coupling (F3165 and F1804). Mag (iron impregnated agarose)-mediated and Ag (agarose)-mediated affinity isolations were performed in parallel (nos. M8823 and A2220, respectively; Sigma-Aldrich). Protein marker (Bio-Rad Precision Plus Protein All Blue Standards 161-0373).

Figure 2. Titration of RRP6-3×FLAG expression

DMEM containing 320 ng/mL tetracycline was serially diluted with DMEM to 5 ng/mL tetracycline, and each dilution added to cells to induce RRP6-3×FLAG expression. Ten micrograms WCE were run on a 6% Tris-glycine gel and transferred to a PVDF membrane. The membrane was probed with antibodies against the endogenous RRP6 protein.

Figure 3. Cryogenic disruption provides lower background purifications

Control HeLa cells or HeLa cells expressing LAP-tagged RBM7 protein were lysed using either sonication or cryogenic grinding and extracted in 20 mM HEPES, pH 7.4, 300 mM NaCl, 0.5% v/v Triton X-100. Clarified cell lysates were subjected to affinity isolation using MBs coupled to anti-GFP antibodies (S, sonication, G, grinding).

Figure 4. Copurification of complexes with 3×FLAG- and LAP-tagged proteins using magnetic beads

Cryogenically disrupted HEK cells (100 mg) expressing RRP6-3×FLAG or RRP41-3×FLAG proteins and HeLa cells expressing RBM7-LAP were used in affinity isolation experiments with MBs coupled to anti-FLAG or anti-GFP antibodies, and eluted with LDS; 200 mg disrupted HEK cells expressing CBC20-3×FLAG were affinity isolated with MBs coupled to anti-FLAG antibodies and eluted with 3×FLAG peptide. All isolations were done in 20 mM HEPES, pH 7.4. Exosome isolations were done at 300 mM NaCl with 0.5% v/v Triton X-100; NEXT complex at 500 mM NaCl, 0.5% Triton X-100; and CBC at 100 mM NaCl, 1.0% v/v Triton X-100. Relevant proteins identified by MALDIMS are indicated. *Ig heavy chain.