Protocols - Protein staining
Colloidal Coomassie Staining
We established a modified colloidal Coomassie staining procedure using Coomassie Brilliant Blue G-250 with phosphoric acid in the presence of ammonium sulfate according to Neuhoff (Electrophoresis 1985, 6: 427-448 and 1988, 9: 255-262). This procedure allows the detection of approximately 10 ng protein per band and is fully compatible with mass spectrometric protein identification. We recommend to perform some test stainings before processing the gels of interest.
Download colloidal coomassie protocol (PDF)
| ||Example 1: 1D-gel|
LMW marker (GE Healthcare
Amount of protein applied (calculated for BSA = 66 kDa):
100 ng, 50 ng, 20 ng, 10 ng, 2 ng BSA (from left to right)
| || |
We have also tested other variants of the Neuhoff staining procedure which have been recently introduced with the objective to further increase the sensitivity of colloidal Coomassie staining. However, in our hands using the test system shown in Example 1, the modifications according to Herbert et al. (Electrophoresis 2001, 22: 2046-2057) and Candiano et al. (Electrophoresis 2004, 25: 1327-1333) were not found to increase the sensitivity significantly in comparison to our colloidal Coomassie staining procedure. The same holds true for fixing the gel with phosphoric acid instead of acetic acid, a modification commonly used in some laboratories.
We use a MS-friendly silver staining procedure on the basis of the widely used Blum protocol (Electrophoresis 1987, 8: 93-99), which comprises modifications introduced by the Görg lab (Proteomics 2001, 1: 1359-1363) and ourselves.
Download silver staining protocol (PDF)
Note that mass spectrometric protein identification after silver staining is more often prone to fail, which may be due to the high staining sensitivity and/or incompatibilities with the subsequent work flow (e.g. formaldehyde treatment). We therefore recommend the use of our colloidal Coomassie staining protocol whenever possible.
In our hands, destaining of the silver-stained gel plugs prior to digestion does not improve protein sequence coverage significantly, which is in agreement with results from other laboratories.
We have also tested noncovalent fluorescent stains such as SYPRO Ruby and ruthenium II tris-bathophenantroline disulfonate (RuBP) known to be more sensitive than colloidal Coomassie stains. On the basis of our test experiments, we concluded that the full potential of SYPRO Ruby and RuBP can only be realized with a CCD camera- or laser scanner-based fluorescence detection. However, since these detection methods are not readily compatible with manual spot picking, a robotic spot picking system is required to take the full advantage from protein staining with fluorescent dyes.
For our test experiments, we used a blue fluorescent light transilluminator (Dark Reader, Clare Chemical Research) to enable the visualization, documentation, and spot picking of SYPRO-Ruby- and RuBP-stained gels without the need of expensive hardware. Thereby, it was possible to photograph the gels with a conventional digital camera and manual spot picking could be performed in a darkened room. Compatibility with mass spectrometric protein identification was confirmed for both stainings. However, when the Dark Reader was used to detect SYPRO-Ruby- and RuBP-stained proteins, staining sensitivity was found to be in the same range but not significantly higher in comparison to our colloidal Coomassie staining procedure.
Berggren, K. et al., Electrophoresis 2000, 21: 2509-2521
Rabilloud, T. et al., Proteomics 2001, 1: 699-704
Lamanda, A. et al., Proteomics 2004, 4: 599-608
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