Fmoc-based Peptide Synthesis Protocol

Rink amide-4-methylbenzhydrylamine (MBHA) resin and fluorenyl methoxy carbonyl (Fmoc)-protected amino acids (Fmoc-Trp(Boc)-OH and Fmoc-Arg(pbf)-OH) were purchased from Novabiochem (Darmstadt, Germany). N,N-dimethylformamide (DMF), N-methylpyrrolidinone (NMP), piperidine, trifluoroacetic acid (TFA) and HPLC grade solvents were purchased from Merck (Germany). 1-hydroxybenzotrizole (HOBt), N,N-diisopropyl carbodiimide (DIPCDI), unnatural Fmoc-amino acids, triisopropyl silane (TIS), crystal violet (CV), glucose, 3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5)), calcein-acetoxymethyl ester (calcein-AM), bovine serum albumin (BSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle medium (DMEM), dimethylsulphoxide (DMSO), vancomycin (VAN), and melittin were purchased from Sigma-Aldrich (USA). The Fmoc derivative of 12-amino dodecanoic acid was prepared using literature protocol^{63 (link)} and was characterized using RP-HPLC and LC-ESI-MS. Cation-adjusted Mueller-Hinton Broth (MHB) and tryptic soy broth (TSB) were purchased from Difco (USA). Brain heart infusion (BHI) medium and agar powder were purchased from Himedia (India). Live/Dead BacLight viability assay kit and Alamar blue (AB) reagent were purchased from Invitrogen (Eugene, OR). pBluescript II SK(+) phagemid kit was purchased from Agilent Technologies. DMF was double distilled prior to use.