Sample Submission
Mass spectrometers are instruments that are extremely sensitive to large range of contaminates such as salts, detergents, and biological buffers. For this reason sample preparation is a critical step for mass spectrometry analysis. For sample submission the nature of the sample and the experiment should be discussed in detail with Majid Ghassemian (mghassem@ucsd.edu) through email communication or in person.
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However, there are some standard protocols that can be followed for SDS_PAGE gel bands.
1) Coomassie or Colloidal blue (preferred) stained gels: Please cut the area of the stained protein band from your gel using a fresh razor blade on a methanol cleaned surface (gloves on). The area should be as close as possible to the protein material and no larger than 1 cm X 2 cm slice. Larger samples will be charged extra due to large volume of gel material. If possible, gel bands should be cut into 1 mmx 1mm cubes and transferred to a new 1.5 ml tube and covered with water. Proteins in gels are highly stable and can be stored at room temp or refrigerated (do not freeze).
2) Silver stained gels: Only Mass spectrometry compatible silver stains (such as Silverquest from Fisher) must be used. Other stains need to be checked to see if they are compatible with mass spectrometry analysis. Please cut the area of the stained protein band from your gel using a fresh razor blade on a methanol cleaned surface (gloves on). The area should be as close as possible to the protein material and no larger than 1 cm X 2 cm slice. Larger samples will be charged extra due to large volume of gel material. If possible, gel bands should be cut into 1 mmx 1mm cubes and transferred to a new 1.5 ml tube and covered with water. Proteins in gels are highly stable and can be stored at room temp or refrigerated (do not freeze).
For Co-IP (Magnetic or agarose or other beads) protein Enriched samples:
If On bead protein digestion and analysis is requested there are critical steps than need to be followed:
1) Ensure that the antibody or other compounds used as bait are cross-linked to the beads.
2) It is critical that Bead/protein complex is washed at least 3 times using water or PBS. The direct digestion of proteins on beads is inhibited by the presence of residual protease inhibitors in the sample and hence the washing is an absolute requirement for mass spectrometry workflows. The experiment will not work without washes.
3) Remove the last wash solution from your sample and freeze your beads for sample drop off or shipping to our facility.
4) Our detection range is a faint silver-stained band so you should test your sample on SDS-PAGE to ensure that the bait protein is viewable using Silver-staining. The higher recovery of bait protein will yield better Co-IP results.
5) A Western signal is NOT an indication that the MS analysis will be fruitful since westerns are far more sensitive than mass spec analysis.
Majid can also help you determine the best type of analysis for your particular question if you are not familiar with what types of questions can be answered by what types of mass spectrometry.
For sample drop off, please see our location to guide you to our facility.