Sample volumes should be less than 150 ul, however with the advent of ABI's Prosorb Sample Preparation Cartridge, sample volumes of up to 750 ul may be sequenced. A minimum of 10 to 50 pmol of sample should be analyzed. Our Procise sequencer can sequence 1-2 pmol of sample at its highest level of sensitivity. However, it is more practical to sequence larger amounts of protein to be confident of the sequence obtained or to be confident that the N-terminus is blocked if no sequence is obtained. In most cases the amount of sequenceable material is underestimated by sample loss, inaccurate quantitation, or N-terminal blockage during sample preparation.
Larger amounts can cause instrument problems.
When submitting electroblotted samples on PVDF for sequencing, always try to have as much protein as you can in as small an area of PVDF as possible. Too much PVDF in the sequencer's reaction cartridge can lead to excessive sequencer background. One reason why the initial yields are often unexpectedly low, is that the amount of sample present is overestimated by the investigator. The most reliable quantitation method is from amino acid analysis. Lowry, BCA, dye binding assays and absorbance are less accurate methods especially in the low microgram amounts.