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. Author manuscript; available in PMC: 2015 Jun 1.
Published in final edited form as: Protein Expr Purif. 2014 Mar 6;98:46–62. doi: 10.1016/j.pep.2014.02.015

Figure 4. Purification of Heterologously Expressed Neuronal Proteins with Endogenous PDZ Domain Ligands.

Figure 4

Six heterologously expressed neuronal proteins with naturally occurring PDZ domain ligands were purified on PDZ Domain Affinity Resins as described in Methods: (A) Purification of MBP-GluN2B Tail; (B) Purification of His-tagged r-synGAP; (C) Purification of CRIPT in the presence or absence of reducing agents (RA); (D) Purification of PDZbh; (E) Purification of cypin and MBP-cypin. MBP-GluN2B Tail and PDZbh were bound to PDZ2 Domain-HaloTag-HaloLink Resin and eluted with 400 μg/ml SIESDV peptide. Note that the ligand density of the PDZ2-resin used for purifications shown in (A) and (D) was 53 pmol/μl resin. Subsequent preparations of PDZ2-resin achieved the higher ligand density of 240-260 pmol/μl resin shown in Table 2. R-synGAP and CRIPT were bound to PDZ3 Domain-HaloTag-HaloLink Resin and eluted with 400 μg/ml YKQTSV peptide. Cypin and MBP-cypin were bound to tandem PDZ1+PDZ2 Domain-HaloTag-HaloLink Resin and eluted with 400 μg/ml SIESDV peptide. In the experiments shown in C and D the resin was incubated with an amount of recombinant POI in excess of the resin capacity. Fractions from each purification and Precision Plus Protein All Blue Standards were separated on a 4-12% gradient SDS-PAGE gel and stained with Gel Code Blue. T, Total protein; S, soluble protein; U, unbound protein; W, wash; E, peptide eluate; C, concentrated peptide eluate.