Tetramic ri peptide mw 4793

Tetramic RI peptide (MW 4793) was synthesized by Lifetein (Somerset, NJ). Conjugation of Tetramic RI peptide and Mal-PEG4 was performed by Biosynthesis (Lewisville, Texas). The conjugation was purified and assayed using LC-MS by Biosynthesis. Successful conjugation was confirmed using ELISA for detection of tetramer peptide levels in the conjugate. On a microplate, different concentrations of conjugated peptide (0, 1, 5, 10 ng) were added in triplicate and incubated for 1 h at 37°C. The microwells were then blocked with 3% gelatin in TBS for 1h at 37°C. Wells were probed with rabbit anti-RI-peptide antibody [12 ] at 1/2500 in TBST for 1h at 37°C, then washed 10 times with TBST. The wells were treated with goat anti-rabbit HRP antibody (1/5000, Invitrogen) for 1h at 37°C. After washing the wells 10 times with TBST, 100 μl HRP substrate solution (SureBlue, KPL, Gaithersburg, MD, USA) was added and incubated for 1min at RT. One hundred microliters of 1N HCl was added to stop the reaction and the absorbance was read on Elx800 plate reader at 450nm.