e1 plus e2  (Addgene inc)

Journal: Protein engineering, design & selection : PEDS

Article Title: Study on CCR5 analogs and affinity peptides.

doi: 10.1093/protein/gzr062

Figure Lengend Snippet: Fig. 1. Models of the CCR5 generated by PDBviewer 4.0. (A) CCR5 model showing domains of ECL1, ECL2 and Nt as magenta ribbons and other parts as gray ribbons. (B) CCR5 model showing Nt, ECL1 and incomplete ECL2 contained in CCR5 mN-E1-E2. (C) CCR5 model showing Nt and complete ECL2 contained in N-Linker-E2.

Article Snippet: Approximately 500 mg of the recombinant protein CCR5 N-Linker-E2 or CCR5 mN-E1-E2 was added into 40 ml of Protein G PLUS-Agarose (Santa Cruz Biotechnology, USA) and 2 mg of primary antibody CCR5 ECL2 mAb 3A9 (purified mouse IgG2a, k isotype, BD Pharmingen) or CCR5 Nt mAb 2D7 (purified mouse IgG2a, k isotype, BD Pharmingen).

Techniques: Generated

Journal: Protein engineering, design & selection : PEDS

Article Title: Study on CCR5 analogs and affinity peptides.

doi: 10.1093/protein/gzr062

Figure Lengend Snippet: Fig. 2. SDS–PAGE (16.5%) of tagged CBD precursors showing expression under various temperature conditions and WB identification. Plasmids pTwin I-CCR5 N-Linker-E2 and pTwin I-CCR5 mN-E1-E2 were transformed into E.coli strains BL21 (DE3). After incubation, the sonicated supernatant and pellet of fusion proteins recovered from equal harvested culture volume were loaded onto each lane. The arrows indicate fusion proteins CCR5 N-Linker-E2-Mxe intein-CBD and CBD-Ssp intein-CCR5 mN-E1-E2 with molecular weights of ≏36.5 and 33.8 kDa, respectively.

Article Snippet: Approximately 500 mg of the recombinant protein CCR5 N-Linker-E2 or CCR5 mN-E1-E2 was added into 40 ml of Protein G PLUS-Agarose (Santa Cruz Biotechnology, USA) and 2 mg of primary antibody CCR5 ECL2 mAb 3A9 (purified mouse IgG2a, k isotype, BD Pharmingen) or CCR5 Nt mAb 2D7 (purified mouse IgG2a, k isotype, BD Pharmingen).

Techniques: SDS Page, Expressing, Transformation Assay, Incubation, Sonication

Journal: Protein engineering, design & selection : PEDS

Article Title: Study on CCR5 analogs and affinity peptides.

doi: 10.1093/protein/gzr062

Figure Lengend Snippet: Fig. 3. Purification and identification of recombinant proteins CCR5 N-Linker-E2 and CCR5 mN-E1-E2. (A) Intein-mediated recombinant protein CCR5 N-Linker-E2 was purified by CBD chromatography (Peak 1, DTT; Peak 2, CCR5 N-Linker-E2; Peak 3, intein-CBD-tag) followed by Sephadex G-25 to eliminate DTT (Peak 1, DTT; Peak 2, CCR5 N-Linker-E2). SDS–PAGE shows the single band from Peak 2. (B) Intein-mediated purification of the recombinant protein CCR5 mN-E1-E2 by CBD chromatography. SDS–PAGE shows a single band from Peak 1 (Peak 1, mN-E1-E2; Peak 2, CBD-intein-tag). (C) and (D) denote purity and molecular weight of N-Linker-E2 and mN-E1-E2, respectively, determined by HPLC and MS analyses.

Article Snippet: Approximately 500 mg of the recombinant protein CCR5 N-Linker-E2 or CCR5 mN-E1-E2 was added into 40 ml of Protein G PLUS-Agarose (Santa Cruz Biotechnology, USA) and 2 mg of primary antibody CCR5 ECL2 mAb 3A9 (purified mouse IgG2a, k isotype, BD Pharmingen) or CCR5 Nt mAb 2D7 (purified mouse IgG2a, k isotype, BD Pharmingen).

Techniques: Recombinant, Chromatography, SDS Page, Molecular Weight

Journal: Protein engineering, design & selection : PEDS

Article Title: Study on CCR5 analogs and affinity peptides.

doi: 10.1093/protein/gzr062

Figure Lengend Snippet: Fig. 4. Immunoassay of recombinant extracellular domains of CCR5 analogs. (A) IP of recombinant proteins CCR5 N-Linker-E2 and CCR5 mN-E1-E2 with mAbs 2D7 and 3A9 by silver staining in 16.5% Tris–Tricine–SDS–PAGE. (B) Comparison of exposure epitopes between recombinant proteins CCR5 N-Linker-E2 and CCR5 mN-E1-E2 by indirect ELISA analysis, showing significant differences between the two CCR5 analogs binding to mAb 2D7 and between the two antibodies binding to N-Linker-E2 with P , 0.05. (C) Competitive ELISA of recombinant proteins CCR5 N-Linker-E2 with Met-RANTES. (D) Competitive ELISA of recombinant proteins CCR5 mN-E1-E2 with Met-RANTES.

Article Snippet: Approximately 500 mg of the recombinant protein CCR5 N-Linker-E2 or CCR5 mN-E1-E2 was added into 40 ml of Protein G PLUS-Agarose (Santa Cruz Biotechnology, USA) and 2 mg of primary antibody CCR5 ECL2 mAb 3A9 (purified mouse IgG2a, k isotype, BD Pharmingen) or CCR5 Nt mAb 2D7 (purified mouse IgG2a, k isotype, BD Pharmingen).

Techniques: Recombinant, Silver Staining, SDS Page, Comparison, Indirect ELISA, Binding Assay, Competitive ELISA

Journal: Protein engineering, design & selection : PEDS

Article Title: Study on CCR5 analogs and affinity peptides.

doi: 10.1093/protein/gzr062

Figure Lengend Snippet: Fig. 5. Reactivity between phagotopes isolated by biopanning and the antigen CCR5 N-Linker-E2 or mNE1-E2 determined by capture ELISA (mean and standard deviation of triplicates), showing positive clones (*) with P , 0.05.

Article Snippet: Approximately 500 mg of the recombinant protein CCR5 N-Linker-E2 or CCR5 mN-E1-E2 was added into 40 ml of Protein G PLUS-Agarose (Santa Cruz Biotechnology, USA) and 2 mg of primary antibody CCR5 ECL2 mAb 3A9 (purified mouse IgG2a, k isotype, BD Pharmingen) or CCR5 Nt mAb 2D7 (purified mouse IgG2a, k isotype, BD Pharmingen).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Clone Assay