N-Terminal Protein/Peptide Sequence Analysis

Protein sequencing is performed on an Applied Biosystems Procise model 492 protein sequencer equipped with an on-line PTH-amino acid analyzer and model 610A data system. Generally, 2 to 10 picomoles of available amino terminus are required to obtain useful sequence data. This represents 0.1 to 0.5 micrograms for a 50 kDa protein. Under normal circumstances 20 to 30 amino acids can be identified when sequencing at least 20 picomole amounts. The higher the molecular weight, the more difficult. Proteins of 150 kD may yield no more than five to ten amino acids and require more protein. The minimum quantity for a protein of this size should be 10 picomoles. Sample purity levels of 90% or better give the best results, although satisfactory results can often be obtained on samples of no greater than 70% purity. Samples can be supplied in an aqueous solution (absolutely no ammonium or TRIS buffer salts), speed-vac dried or electrotransferred to PVDF membranes. Proteins bound to PVDF may be stained by Coomassie Blue R-250, Ponceau S, or Amido Black. The stained protein band can be excised with a clean razor blade (wear gloves to avoid contamination of blot) and placed in a 1.5 ml conical centrifuge tube. The protein is stable for at least 6 months when stored at -20 degrees C. The electrophoresis step should be performed on a gel polymerized for a minimum of 18 hrs to avoid N-terminal blockage by residual acrylamide monomer. Even with these precautions one should assume a recovery of no more than 25-50% of the protein (N-terminus) after electrophoresis and electroblotting to PVDF.

Liquid samples should have a maximum volume of 90 microliters. Suitable solvents include: water, various concentrations of acetic acid, or trifluoroacetic acid, or aqueous solutions with up to 0.1 M (in 90 microliters) salts or buffers such as NaCl, phosphate, HEPES, MOPS, MES, etc. If necessary, up to 0.1% sodium dodecyl sulfate (per 90 microliters volume) may be used, but no other detergents are compatible. Dried samples should include the recommended solvent for reconstitution for application to the sequencer.

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  • $100 setup charge per sample, plus $18 per amino acid cycle (five cycle minimum)