acknowledges the Foerderer Scholar Fellowship Award, the Dubbs Scholar Fellowship Award, the Measey MD/PhD Student Fellowship, and Ruth L. L fragments), 4HJG (memAb trastuzumab with protein A and L fragments), and 4IOI (memAb trastuzumab with cQFD and protein A and L fragments). Analytical SEC. Ten micromolar cetuximab Fab, EGFRdIII, and 5 M meditopeCFc were DMT1 blocker 2 used for the individual runs and for the binary complexes. The complex of the three components was formed with stoichiometric complex of the Fab and EGFR added to the meditopeCFc. The proteins were mixed and incubated at room temperature for 20 min and applied to a Superdex 200 Rabbit polyclonal to AGAP9 10/300 column (GE Healthcare) at 4 C. Similar procedures were used with the parental or meditope-enabled trastuzumab IgGs, sHER2 and meditopeCFc. BLAST Search. Using the nonredundant database and DMT1 blocker 2 filtering for em Homo sapiens /em , we used the cetuximab and trastuzumab sequences as search sequences and aligned the top 1,000 sequences returned from each. A multisequence alignment of light chain residues 26C89, starting with cetuximab (1YY8_A), is presented as Alignment S1 with Thr40, Asn41, and Asp85 highlighted in magenta. SPR Binding Experiments. All SPR experiments were performed on a GE Biacore T100 instrument at 20 C, and analysis was performed by using Biacore T100 Evaluation software version 2.0.1. Ligands were amine coupled to CM5 chips at low densities suitable for kinetics. Concentrations of analytes were prepared in HBS-EP+ buffer (10 mM Hepes at pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% vol/vol surfactant P20), which was used as a running buffer in all experiments. For saturation analysis, cQFD meditope or EGFRdIII affinities to immobilized cetuximab scFv or Fab were assessed by equilibrium methods at 20 C and fit to the equation: RU = (RMAX ? [L])/([L] ? em K /em d) + ROFFSET. Supplementary Material Supporting Information: Click here to view. Acknowledgments We thank Dr. Stephen Gillies for providing us the CH14.18 sequences. J.M.D. acknowledges the Foerderer Scholar Fellowship Award, the Dubbs Scholar Fellowship Award, the Measey MD/PhD Student Fellowship, and Ruth L. Kirschstein National Research Service Award T32 DK07705; and support from the Alicia and John Kruger Gift, the Nesvig Foundation, and National Cancer Institute (NCI) Grant R21 CA135216. C.Z. acknowledges support from the Gastrointestinal Cancers Program Pilot Grant at City of Hope. The project described was also supported by NCI Grant P30 CA033572. Footnotes Conflict of interest statement: D.A.H. and J.C.W. have founded Meditope Biosciences, a company based on some of the observations presented here. *This Direct DMT1 blocker 2 Submission article had a prearranged editor. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 4GW1, 4GW5, 4HKZ, 4HJG, and 4IOI). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1307309110/-/DCSupplemental..