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Edman Sequencing Sample Protocols

Sample Attributes for Successful Edman Sequencing

Sample Purity
Amino-terminal heterogeneity makes interpretation of Edman chromatograms difficult. As a general rule we suggest that soluble proteins for sequencing be more than 80% pure.

Sample Buffer
The sample should be in a volatile solvent such as H2O, acetic acid, acetonitrile, propanol, formic acid, TFA, TEA, or a volatile buffer such as ammonium bicarbonate. Detergents are particularly problematic in sequencing and should be avoided if possible. Small quantities of detergent may be used (i.e., 0.02% Tween 20, 0.01% Brij 35, 0.1% SDS, 0.05% Triton X 100). All reagents used to resolve and solubilize proteins should be of the highest quality. Reagents that contain primary amines (Tris, glycine, ethanolamine) should be avoided. In addition, it is important to note that dialysis membranes are often a source of interfering contaminants. If dialysis is necessary in the purification process, only high quality, thoroughly cleaned membranes (e.g.,Spectropor) should be used.

When samples are transferred to PVDF membranes many of these difficult reagents can be washed away with water and 50% methanol.

Sample Amount and Volume
Our ABI Procise sequencer is capable of sequencing sub-pmole amounts of peptide but we recommend that a minimum of 10- 50 pmoles of sample be sent for analysis. This will allow us to ensure that sequence interpretations are correct and to determine if the amino-terminal is blocked if no sequence is obtained. Sample quantity can be estimated from Coomassie Blue or silver-stained gels or determined by a number of protein quantization assays. Sample loss is often caused by the adsorption of protein to purification apparatus and storage containers. To avoid this problem, try eliminating as many handling steps as possible. Use polyethylene or polypropylene micro centrifuge tubes keeping protein mass to plastic area as high as possible and avoid drying or lyophilizing samples completely.

Tips for SDS Gel Electrophoresis Prior to Sequencing

  • Use ultra-pure reagents.
  • Make up stock acrylamide solution fresh every 2 to 3 weeks.
  • De-ionize the acrylamide stock solution with ion exchange resin and store it in a brown glass container in a cool place.
  • Polymerize gel overnight to minimize any amino-reactive free acrylamide.
  • Pre-run the gel (5-10 minutes) before loading your sample.
  • Denature sample for 0.5-1hr at 65C - avoid boiling sample.

Electroblotting, Polyvinylidene Difluoride (PVDF)

  1. Prepare CAPS buffer.
    • 10 x stock (100 mM, pH 11). Dissolve 22.13 g of 3-[cyclohexylamino] 1-propanone-sulphonic acid in 990 ml of de-ionized water. Titrate with 2 M NaOH (~ 15 ml) to pH 11 and add de-ionized water to make final volume of 1 L. Store at room temperature. CAPS (Sigma C2632).

    • Electroblotting buffer. Prepare 2 L of buffer by mixing 200 ml of 10 X stock buffer, 200 ml of methanol, and 1600 ml of de-ionized water.

  1. Wet the high-binding PVDF membrane (Millipore Immobilon PSQ, ABI Problott, Pall Fluorotrans, BioRad transblott or any other sequencing grade PVDF membrane) with methanol for a few seconds and place them in a dish containing blotting buffer.

  2. Remove the gel from the electrophoresis cell and soak in electroblotting buffer for 5 minutes.

  3. Assemble the transblotting sandwich and electroblot at 30 V overnight or at 100 V (1-2 hours) with a cooling unit (for mini-gel). Since the transfer is protein dependent, use your preferred condition.

  4. Remove the membranes and rinse with de-ionized water prior to the staining.

Detection

Protein blotted onto PVDF membranes can be visualized by two different staining procedures with either Coomassie blue or Ponceau S.

  1. Following electroblotting the membrane needs to be rinsed in de-ionized water for 5 minutes. If you use Coomassie then saturate with 100% MeOH for a few seconds.

  2. Coomassie blue R-250 (0.1% solution in 40% methanol/10% acetic acid) staining is carried out for 5 minutes followed by de-staining (5-10 minutes) in a methanol solution (50% methanol).

  3. Ponceau S (0.2% solution in 1% acetic acid) staining is carried out for 1-2 minutes followed by a simple rinse with de-ionized water.

  4. We will excise the bands from the stained blot