Reverse Phase HPLC
Automated peptide mapping by C-18 reverse-phase HPLC is available on a HP1090 system that has been optimized for microbore HPLC (1 x 250 mm columns). Digests are loaded in 50 μl of a suitable solvent, (which should not contain detergent) and should contain from 5 to 100 pmol protein. Unless otherwise requested, peptides will be detected by their absorbance at 210 nm.
Protein digests that contain from 5 to 100 picomoles in a volume of 50 µl may be fractionated by C-18 reverse-phase HPLC and automatically collected with peak detection into 100-150 individual Eppendorf tubes. If further purification is required, individual peaks collected from a HPLC collection run of a protein digest can be re-chromatographed on an Aquapore C-8 column. Cerenkov counting, which can detect high energy beta emitters like [32P] without having to add scintillation fluid to the sample, of up to 100 HPLC fractions from a single collection run is also available.
Intact proteins are fractionated on a non-porous reverse phase column (normal TFA buffer system) on the 2nd dimension of the Beckman-Coulter ProteomeLab PF2D system. 50μl is injected from an appropriate sample buffer of 2M urea, 50 mM Tris, and 6.25mM TCEP. The recommended minimum amount is 200μg.