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Protein Sequencing Sample Submission Guidelines

 
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04/26/2009

Protein Sequencing Sample Submission Guidelines

Protein Sequencing Sample Submission Guidelines

Bio-Synthesis offers both N-terminal and C-terminal sequence of protein and peptides by the Edman procedure.  Edman degradation is a cyclic procedure where PITC is coupled to the N-terminal amino group followed by cleavage of the N-terminal residue. The PITC coupled residue is transferred to a flask, converted to a PTH-residue and analysed by injection on an HPLC column.

 The standard service includes analysis of the first six (6) amino acids (6 cycles). Please indicate on sample submission form if additional cycles are requested. The analysis can provide up to 20 amino acids.

To read more about the Bio-Synthesis Molecular Weight Determination Analysis  at www.biosyn.com.

Samples can be submitted in liquid form, solid form or as spots/bands from PVDF membranes.

PVDF membrane samples

  1. Run your 1D/2D gel
    1. Run 1D or 2D gel
    2. Blot onto PVDF membrane – DO NOT use Nitrocellulose membranes!
    3. All handling should take place in a dust free environment
  2. Stain the PVDF membrane with Coomassie Blue or Ponceau staining
    1. Coomassie Blue and Ponceau works well in combination with N-terminal Edman Sequencing – DO NOT use silver staining!
    2. Wash PVDF membrane several times in water
    3. Notice that glycine in the transfer buffer requires very extensive washing
  3. Cut out the bands/spots of interest
    To be able to obtain good sequencing data you should only cut spots or bands that are clearly visible by Coomassie Blue/Ponceau staining
    1. Use a clean scalpel
    2. Cut out the band as close to the protein edge as possible to have the protein concentration high in the smallest membrane volume possible
    3. Clean the scalpel in-between spots/bands
    4. Please supply
  4. Put the bands/spots into separate wells/tubes
    1. Use the Eppendorf Safe Lock tube supplied with the kit or similar tubes from your lab

Liquid samples

  1. Purity and sample amount
    1. The chromatographic protein purity should be >90%
    2. Avoid all detergents and keep buffer concentration at a minimum
    3. Avoid contamination of the sample with dust, i.e. human keratin d . Minimum amount ~ 1-10 micrograms
  2. Put the protein/peptide into microcentrifuge tubes
    1. Use the Eppendorf Safe Lock tube.
    2. Lyophilize or speed-vac the protein/peptide to a solid sample to ensure protein stability during shipment
    3. Alternatively, refrigerate to +4oC for cold shipment of liquid
    4. If the sample requires special treatment to bring into solution (e.g. organic solvents, extensive ultrasonic bath etc.) please advise

Sample Shipment

The sample is now ready for submission

  1. Download, fill out and sign the Sequencing Sample Submission Form
  2. Place the tube(s) in the bubble bag to protect the tube during shipment.
  3. Ship the samples using a courier company. Liquid sample should be shipped at +4oC and gel samples at ambient temperature. Ship to:

    Bio-Synthesis Inc,
    Proteomic Analytical Division
    612 E. Main Street,
    Lewisville, TEXAS, 75057
    USA
    800.227.0627 (toll free)
    972.420.8505 (oversea)
    972.420.0442 (fax)