Research Technologies Branch

Protein Chemistry

Protein Sequencing: Protocols

Sample Preparation

There are two ways to submit a sample for sequence analysis:

1. Soluble protein or peptide sample (in sequencer compatible reagents)
2. Protein/peptide sample blotted onto polyvinylidene difluoride (PVDF)

Soluble Sample Preparation Guide

• Volumes for soluble samples should be kept under 0.5ml. HPLC or cLC offer good resolution, and put the sample in sequencing compatible solvents (TFA/water/acetonitrile). Avoid Tris, guanidine, glycerol, sucrose, ethanolamine, SDS, Triton X-100 (and other detergents), and ammonium salts.
• HPLC fractions can be rapidly screened by MS (require <1 picomole), before committing to sequence analysis.
• Purity is of utmost importance. Work in low traffic areas whenever possible. Avoid skin contamination (amino acids, keratins).
• The soluble sample can be applied to a polybrene-treated glass fiber filter, or applied to PVDF using a ProSorb device (ABI). Sometimes sample contaminants can be washed away by using the ProSorb device.

Samples Blotted onto Polyvinylidene Difluoride (PVDF)

• PVDF is available from a variety of vendors (Immobilon, Millipore; ProBlott , ABI). A good procedure for electroblotting onto PVDF is described in chapter 10 of Current Protocols in Protein Science. Pre-run gels to avoid N-terminal blockage. Samples may be run on 1D or 2D SDS-PAGE. One "spot" on an SDS gel rarely means that only one protein is present. Better resolution is achieved with 2-D gel electrophoresis. Use thin gels for better efficiency of transfer. Protein blotted onto PVDF can be visualized by staining with Coomassie blue, amido black, Ponceau S, or sypro orange. A general rule of thumb is that "if you can see a Coomassie stained gel spot, it can be sequenced." Avoid colloidal metals like silver or gold.
• PVDF strips will be directly applied to the reaction cartridge of the sequencer. Approximately 50 square millimeters of PVDF can be used. Thus several cut out strips, or sample runs, can be applied per sequencing run.
• The cut out band(s)/spot(s) should be stored in clean tubes, and are stable for shipping at room temperature. The affinity of peptides and proteins to PVDF membranes is structure dependent. The more hydrophobic the peptide, the more affinity it has for the PVDF membrane.

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