The instruments deliver precise volumes of reagents to a cartridge where the polypeptide is immobilized on either an acid-etched glass filter coated with polybrene or a PVDF membrane. At each cycle the derivatized amino acid is transferred to the HPLC for analysis and quantification. In most cases, picomoles required for sequencing. It is possible to obtain sequence data on as little as 1-2 picomoles of clean peptide or 5 picomoles of clean protein. The amount of protein or peptide necessary to obtain unambiguous sequence is dependent on the sample.
Experience indicates two main problems with incoming samples which are analyzed.
The second problem is that investigators usually over estimate the amount of protein in the sample by as much as ten-fold. Both of these problems need to be addressed before a sample should be placed on the sequencer. In the preparation of samples for sequencing the investigator should be cautioned that at this level of polypeptide, losses can occur when transferring samples from tube to tube. Many times the samples are lost due to adsorption onto plastic or glass. To prevent such loss, the investigator may wish to adsorb the protein on to PVDF membrane.