Protein Identification by MALDI-TOF/TOF

Sample Info for gel bands (1D) or spots (2D)

Please keep in mind that a single band from a 1D gel could contain several proteins. This can reduce the real amount of your protein of interest. Low quantity and high complexity of samples will greatly reduce the chance of success by MALDI-TOF/TOF. In general, MALDI-TOF/TOF is ideal for identifying a single protein from 2D gel spots. Please follow the tips below:

Protein amount: Our general rule of thumb is that if there is enough protein to be visible with Silver Staining or Coomassie G-250, there should be enough for Mass Spectrometry analysis. We prefer that you scale up your prep so that you have 10-20 ng of protein in the gel band. Please do not scale up by sending multiple gel pieces, as this increases gel volume and decreases the efficiency of peptide extraction from the gel. Instead, load a higher amount of protein into ONE lane of the gel.

Minimize gel volume. It is important to minimize the gel volume and maximize protein concentration. As such, we recommend you cut out only the area with your protein of interest and exclude as much unstained gel as possible.

Silver stained gel band should be compatible with Mass Spectrometry. You need to make sure your silver staining solution does not contain aldehyde that cross-links your proteins to the gel matrix, resulting in low extraction efficiency. Non-formaldehyde Silver Stain uses carbohydrazide instead of formaldehyde for reduction/development.

Shipping condition. When you are ready to send your sample, please add HPLC-grade water to cover the excised gel band in a 1.5ml eppendorf tube, or 96 well plate. Seal the Eppendorf tubes with Parafilm. Put the Eppendorf tubes into 50 mL conical tubes, then pack the 50 mL tube with Kimwipes to prevent the Eppendorfs from being crushed or jostled. The samples can be shipped on ice pack (no need to send on dry ice).