Protein and Peptide Sequence Analysis
The PSCF has an ABI Procise 494 Sequencer. It is used to determine the amino acid sequence of proteins or peptides by the Edman procedure. The instrument delivers 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. 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 three main problems with incoming samples which are analyzed. The first is the number of proteins presented to core facilities having a blocked N-terminus, thus preventing N-terminal sequencing of the protein itself. The second problem is that investigators usually over estimate the amount of protein in the sample by as much as ten-fold. The third is a higher level of free amino acids and other proteins present on the sample. All 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. Samples can be blotted directly onto PVDF from polyacrylamide gels using standard techniques. (For copies of the protocol please call the PSCF.) Problott cartridges are also available from the facility for concentration of proteins and separation from unwanted buffers and salts. Samples once dried in a tube may not be easily dissolved, so other options should be investigated.
- Nebraska Prostate Cancer Research Program (NPCRP)
- Confocal Laser Scanning Microscope
- Epigenomics Core Facility
- Protein Structure Core Facility (PSCF)