Sample Preparation Guidelines

Services Protein in Solution / Solid  Gel Slice from SDS PAGE  PVDF membrane
Protein identification by MS - MALDI-TOF/TOF
  • Pure protein: 100ng to 1ug, <250uL
  • Best to freeze-dry or air-dry in 1.5mL tube
  • Free of interfering buffers and salts (Client must declare what buffers and salts were used prior to sample drying.  If a result of extraction, it will help to provide extraction procedure, eg HPLC, Protein preciptation, MWCO.)
  • NOTE:                                             
    Buffers containing primary amines, such as TRIS and HEPES, should be removed. Low concentrations of SDS (up to 0.3%) and PBS can be tolerated. There may be other interferring buffers not common to these, thus always provide buffer composition prior to drying.
  • De Novo service, same as the above         
  • De Novo service, Pure protein: Normally 10ng-100ng. Again, for freeze-dried or powdered: 50µg 
  • Yes. Cut into 1.5mL tube and dried.

    NOTE: no water or buffer!
     
  • Minimum 2 spots: Commassive Blue; or MS compatible Silver Stain (1 month). (Each spot contain minimum 100ng)
     
  • Minimum 4 spots: SYPRO Ruby; or Cy dyes.                                                               MALDI-TOF/TOF: Sample size >20kDa LC/MS/MS: sample size <20kDa
     
  • De Novo service, same as the above 
Never
Protein identification by MS - Electrospray (LC/MS/MS) 
De Novo Protein Sequencing 
iTRAQ analysis 
  • 200-500µg of sample                   
  • Cell lysates, secreted protein samples, but not radioactive samples.                                    
  • Ship sample with dry ice  
    NOTE: Please make sure that all serum or related culture medium are washed off before lysing cells. For any questions, please write-in to enquire.
Never Never
Protein Mass analysis
  • Pure protein, must be freeze dried from low salt buffer.      
  • The protein concentration before drying must be at least 0.1mg/ml.
  • The solution must be a low salt buffer, e.g. 50mM ammonium hydrogen carbonate; for TRIS or phosphate buffers the maximum salt strength is 20mM. NaCl and surfactants (e.g. SDS) cannot be present.
Never Never
Amino Acid Analysis (AAA)
  • The minimum amounts are:
    High sensitivity AAA – 10 μg (5μg in duplicate)
    Cys – 20μg (10μg in duplicate)
    Trp – 1mg (0.5mg in duplicate)
  • Sample must be freeze dried.   
  • Information needed:
    (1) The volume of the liquid before drying.
    (2) Solvent/buffer used before drying.
    (3) Known or estimated concentration of sample.
Never Never
N-Terminal Protein Sequencing 
  • Diluting the sample with a small amount (25 ul for up to 100 ug of protein) of SDS boiling buffer without reducing agents (5% SDS, 10% glycerol and 60 mM Tris, pH 6.8) and freeze drying by lyophilization.
  • Solution sample: with dry ice shipment. (Additional charges apply for run gel & PVDF membrane blotting services)
Never
  • Should be stained with
    Coomassie Blue, and the target band must then be visible by eye. Blots must be free of any particulate material.
  • Attach gel photo to indicate the exact band to be cut (by us) and sequenced.
Photos

 


NOTE: 
Please be reminded that to avoid evaporation, leakage or lost of samples, all sample tubes MUST be wrapped with para-film before shipping them to our lab.