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  • Protocol
  • Published:

Photo-cross-linking interacting proteins with a genetically encoded benzophenone

Nature Methods volume 2, pages 377–384 (2005)Cite this article

  • Talon metal affinity resin (BD Biosciences)

  • Coomassie (Bradford) Protein Assay kit (Pierce)

  • E. coli strain DH10B, prepared as electrocompetent cells

  • Elution buffer: 50 mM sodium phosphate, 300 mM sodium chloride, 150 mM imidazole (pH 7)

  • Heavy metal–containing glycerol minimal medium10 prepared as described in the text (Step 5)

  • InVision His-tag In-gel stain (Invitrogen)

  • Lysis buffer: 1 mg/ml lysozyme, 50 mM sodium phosphate, 300 mM sodium chloride (pH 7)

  • PAGEr Gold precast polyacrylamide gels, 4–12% Tris-glycine, 9 × 10 cm, 1-mm thick (Cambrex)

  • Phosphate-buffered saline (PBS): 10 mM sodium phosphate, 140 mM sodium chloride (pH 7.2)

  • pBpa (Fig. 1a; Bachem) solublized in 1 M sodium hydroxide solution (Step 6)

  • Petri dish (polystyrene, tight lid, 47-mm; Fisher)

  • Plasmid vectors: pTrcHis (Invitrogen); pDULE-pBpa (Fig. 1b),

  • pDULE-Tyr (Fig. 1b; available on request from Peter Schultz (Schultz@scripps.edu)

  • Poly-Prep chromatography columns (0.8 × 4 cm; Bio-Rad)

  • Polystyrene 96-well plate (flat, low evaporation, 0.1–2 μl; Costar)

  • Selective medium: 2× YT

  • SOC medium

  • Wash buffer: 50 mM sodium phosphate, 300 mM sodium chloride (pH 7)

  • Isopropyl-β-D-thiogalactoside (IPTG)

  • PD10 columns (Amersham)

Talon metal affinity resin (BD Biosciences)

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Figure 1: Structure of pBpa and a schematic of the pDULE plasmid.
Figure 2: The steps to express a protein with a site-specifically incorporated pBpa in E. coli.
Figure 3: Analysis of pBpa-containing protein production.
Figure 4: Irradiation time courses of HDH-225pBpa and HDH-225Tyr.

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Acknowledgements

J.W.C. and R.A.M. thank Peter Schultz for his support in the development of some of the vectors described in this protocol.

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Authors and Affiliations

  1. Chemistry Department, Franklin and Marshall College, P.O. Box 3003, Lancaster, 17604, Pennsylvania, USA

    Ian S Farrell, Rebecca Toroney, Jennifer L Hazen & Ryan A Mehl

  2. MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK

    Jason W Chin

Authors
  1. Ian S Farrell
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  2. Rebecca Toroney
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  3. Jennifer L Hazen
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  4. Ryan A Mehl
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  5. Jason W Chin
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Correspondence to Ryan A Mehl or Jason W Chin.

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Farrell, I., Toroney, R., Hazen, J. et al. Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Nat Methods 2, 377–384 (2005). https://doi.org/10.1038/nmeth0505-377

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