Biotinylation of tyramine. Biotinyl-6-aminohexanoic Fludarabine Phosphate NHS ester was dissolved in 50 mM borate buffer (pH cool and tyramine HCl added. The mixture was kept on agitation overnight and filtered through a 45 μM syringe filter. The biotinylation of tyramine was verified by MALDI-MS and blotting analysis. 10% (v/v) DMSO was added upon usage.
pH-dependence of GGH-C-peptide nickel complex formation.GGH-C-peptide was diluted in ammonium acetate buffer, pH 4–8, Ni2+ was added in three-fold molar excess, and samples were incubated for 30 min on ice. Samples were introduced at a flow rate of 5–10 μL/min into an LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, USA), operated in negative ion mode with data acquisition over a mass range of m/z 150–2000.
Labeling of a C-peptide antibody. Preparations of GGH-fused C-peptide, or GGH-tag only, and antibody was activated and allowed to react as described above. A monoclonal anti-C-peptide antibody was used to establish the protocol (Medix Biochemika, Kaunianen, Finland) and a randomly chosen antibody (anti-LL37, Inovagen, Lund, Sweden) was added under the same conditions to verify specificity of the biotin-labeling.
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