Instrumentation

Automated Edman (N-terminal) sequencing is performed with a Hewlett Packard G1005A automated protein sequencer. The system is a complete, automated chemistry and analytical system for the amino acid sequence analysis of proteins and peptides. Composed of a G1000A sequencer, system controller and on-line PTH-amino acid analyzer, the G1005A is capable of routine analysis of dry, liquid or electroblotted protein samples. The procedures are sensitive in the picomole to nanomole range. The instrument has four column modules that are run back-to-back in sequence allowing for continuous operation with a minimum of backlog due to sample loading resulting in a shortened turn-around time for results. A unique feature of the Hewlett Packard system is the biphasic sample-column support technology used for sample application. Using miniature sample-loading columns packed with reverse phase carbon-bonded resins we are able to analyze samples that contain a wide variety of matrix components including buffers, salts, chaotropic agents, reducing agents and even small amounts of detergents. In addition to allowing the removal of contaminants, the biphasic column concentrates the sample allowing the analysis of relatively large volumes of dilute protein samples. The high binding capacity of the columns as well as the tight binding affinity of most proteins and peptides to the resin allow for relatively high initial and repetitive yields. Modified versions of the miniature columns also allow us to sequence proteins that are electroblotted onto hydrophobic membranes (PVDF; polyvinylidenedifluoride) as well as synthetic peptides that are attached to beads.