Sample Submission Information
The sample submission form for gel electrophoresis and mass spectrometry are available here:
Silver based staining methods are not recommended for peptide mass fingerprinting (PMF) due to low yields of tryptic fragments recovered after in-gel digestion. Coomassie based staining has shown to have the lowest impact in in-gel digest reaction, and, therefore, gives the best mass spectrometry results. Gels visualized with Sypro Ruby (fluorescence stain) and CyDye™ (fluorescence-tag labeling) are more sensitive than Coomassie stain, and are compatible with mass spectrometry. However, they are also much more expensive, and may also detect proteins that cannot be analyzed with mass spectrometry (because of differing sensitivities of mass spectrometry and fluorescence analysis). We encourage the investigator to discuss these issues with the Staff to decide the best visualization method for your need.
Gel Electrophoresis Analysis
2D gel electrophoresis samples are ideally:
- < 10 mM salt and buffer (ideally no salt or buffer)
- Avoid using ionic components (such as SDS)
- Can contain 4 % CHAPS and 8 M urea (the IPG sample loading buffer)
- Concentration: 5-10 mg/ml
- Amount: total 50-100 µg of protein extracts.
If you wish to submit raw samples, or questions about sample preparation for extraction, please contact Jesse Guidry.
For assuring the successful analysis, we estimate the minimum amount of a protein sample for successful mass spectrometry analysis:
2D gel spot: ~1 ng or more per spot
1D gel band: ~1 ng or more per band, such as immunoprecipitation isolated protein bands.
Protein complex: ~ 1 µg may be required from these samples isolated from Tanden Affinity Purifcation (TAP) or similar methods. A quick run of 1D gel electrophoresis can help to estimate the concentration and complexity of the samples.
A rule of thumb is that it is likely identified if the band or spot can be observed in Coomassie. Often, the faint spots visualized with Sypro Ruby or CyDyes may not yield good mass spectrometry results due to the much lower detection limit of fluorescence imaging methods (sub ng) than the current mass spectrometry analysis (~1ng). If you run your gels in your lab and cut up your own spots, fresh-prepared gel plugs (not destained) of the same proteins in tubes are preferred. It is better that you harvest the whole protein spots/bands. If you need to image your gel and pick up the spots of interest, please schedule an appointment for the Core personnel to coordinate the experiments. To ensure protein identifications of your samples, you need to denote the origins (taxonomy) of your samples in the sample submission form.
Direct molecular weight measurement of intact protein (larger than10K Da)
Larger quantity is required, at least several µg of protein.
Modification identification (posttranslational or chemical)
~100 ng-1µg of purified protein.
The dynamic range of modified and unmodified copies usually is high (100-fold and higher). Higher quantity usually increases the sequence coverage and the chance to detect modified peptides. A rough estimate of the percentage of the modified protein and possible modified AAs are highly helpful. More information is provided, better chance to detect the modification(s).
Peptide structure verification
~50 ng. The sequence of the expected peptide is needed for verification. True de novo sequencing for totally unknown peptides is not applicable.
The Proteomics Core Facility is located in the Clinical Science Research Building room 331. For further information please contact Jessie Guidry, (firstname.lastname@example.org)