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Proteomics Core

Sample Submission Information

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.

2-D Gel Electrophoresis Sample Submission Form

Sample solution is suggested in buffer:
*< 10 mM salt and buffer (ideally no salt or buffer)
* Avoid 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 volume of 185 µl and 20-200 µg for 11 cm strips.

If you wish to submit raw samples for extraction, please contact Proteomics Core Facility staff for further information.

For further questions about sample preparation, please contact Proteomics Core Facility Staff. The Protocols also contains useful sample extraction protocols for sample extraction.

Mass Spectrometry Sample Submission Form

For assuring the successful analysis, we recommend the amount of a protein sample for mass spectrometry analysis is more than 50 ng per sample (1D gel band or 2D gel spot). ~10 – 50 ng per gel sample may be analyzed by MS and yield an acceptable result. 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 than the current mass spectrometry analysis. 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.

For direct molecular weight measurement of intact protein (larger than10K Da), larger quantity may be required, at least several µg f protein is often required.

The Proteomics Core Facility is located in the Clinical Science Research Building room 331. For further information please contact Chau-Wen Chou, PhD (cchou@lsuhsc.edu).