Tryptic Digestion and Peptide Separation
Some of the proteins presented to core facilities for sequencing are N-terminal blocked. To obtain sequence information from such blocked proteins, prior enzymatic digestion or chemical cleavage is needed to prepare protein fragments for sequencing. The facility offers a variety of options, including tryptic digestion in solution followed by separation of the peptides, and deblocking by several procedures. For such a study, 0.1 nanomoles to several nanomoles of protein are needed.
We also provide in-gel or on-membrane tryptic digestion of proteins. This procedure has been used with as little as 50 picomoles of protein. Digestion can be performed by the PSCF or in the patron's laboratory. The peptides generated can be separated on the facility's HPLC. Analysis of radioactively labeled phosphoproteins is possible with a combination of tryptic digestion, separation of peptides and special sequencing techniques. Sites of phosphorylation can be definitively determined if enough protein is present.
The facility has a Michrom MAGIC HPLC with a photo-diode array detector to separate and analyze peptides for sequencing. Peptides obtained from the HPLC can generally be loaded directly onto the sequencer.
Please include a blank section of gel or PVDF membrane with your sample.
- Nebraska Prostate Cancer Research Program (NPCRP)
- Confocal Laser Scanning Microscope
- Epigenomics Core Facility
- Protein Structure Core Facility (PSCF)