Protein Identification

Sample Preparation Guidelines

Protein identification by mass spectrometry requires a pure protein in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

Sample Preparation

Proteins from 1D SDS PAGE and 2D PAGE gels can be submitted for protein identification analysis.

1. Run your 1D/2D gel

   1. Avoid fingerprints – wear gloves, without talcum powder
   2. Use clean glassware and staining trays
   3. All gel handling should take place in a dust free lab. Dust contains human keratin!
      
      

2. Stain the gel with Coomassie Blue, SYPRO Ruby or Silver stain

   1. Coomassie Blue and SYPRO Ruby works well in combination with mass spectrometry
   2. Silver stain – It is essential to use a staining protocol or kit that has been optimized for MS analysis. We recommend using one of the following kits:
      1. ProteoSilver Plus, Sigma (Product # PROTSIL1 or PROTSIL2)
      2. Dodeca Silver Stain, BioRad (Product # 161-0481 or 161-0480)
      3. Or use the Alphalyse silver staining protocol
         
         
      Do not destain silver-stained samples!
      
      

3. Cut out the bands/spots of interest

   1. Use a clean scalpel and tweezers
   2. Cut out the gel band close to the stain edges to have the highest protein concentration in the smallest gel volume. Ideal band size is 2 x 5 mm.
      Do not cut the gel sample into tiny, tiny gel pieces!
   3. Clean the scalpel in-between spots/bands
      
      

4. Put the samples into separate wells/tubes with water

   1. Use the 96-well plate or Eppendorf Safe Lock tube supplied with the PICK 'n POST kit, or similar tubes from your lab
   2. Add 100-200 μl distilled water to keep each gel plug wet
   3. Use the rubber lid for the 96-well plate. Make sure the lid is properly closed