Protein Chemistry
Protein Sequencing: Technology
Instruments
Applied Biosystems 494cLC procise protein sequencer (2) with four cartridge capability.
Both instruments are automated protein sequencers with online capillary liquid chromatography detection systems. Soluble samples, or samples blotted to polyvinylidene difluoride (PVDF), may be used for analysis. The sensitivity of these instruments is 50 to 100 fmoles. These instruments are used primarily to identify unknown samples and have sensitivity comparable to MS.
Applied Biosystems 494 HT protein sequencer with four cartridge capability.
This is an automated protein sequencer with online microbore LC detection. Soluble samples or samples, blotted to PVDF, may be used for analysis. The sensitivity of this instrument is 0.1 to 1 pmol. This instrument is used primarily for "quality control" or sequence verification.
Applied Biosystems 173A microblotter capillary LC system.
This is a fully integrated capillary LC sample preparation system that isolates and collects small amounts of sample in one step. As little as 500 fmol of sample may be prepared. Protein or peptide fractions collected with the 173A may be sequenced directly from the PVDF. The instrument can separate peptides that SDS gels cannot. If a protein is N-terminally blocked, or an internal sequence is needed, a gel plug containing the compound in question is subjected to enzymatic (e.g., trypsin) or chemical (e.g., CNBr) treatment, and then the peptides are eluted from the gel slice. They are then separated and blotted to PVDF by the 173A, enabling sequence analysis. N-terminal modification or blockage may be due to a formyl group, acetyl group, or other acyl grouping, or to the conversion of an N-terminal glutamine to pyroglutamine.
back to top