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Proteomics/Mass Spectrometry

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Frequently Asked Questions

Q: What kind of service does protein identification include?
A: We provide a protein identification using Q-Tof 2 LC-/MS/MS. This includes in-gel digestion, LC-MS/MS analysis, database searching, and report generation (electronic versions).

Q: What is the turn-around time?
A: The typical turn around time is around 3- 5 days for protein identification from gels. For 2D-LC-MS/MS analysis and 2D gel electrophoresis, it takes about one week to complete the whole procedure.

Q: What happens if we send CFG a sample for analysis and for whatever reason, CFG could not generate any data?
A: If no data is obtained we will charge ONLY the in-gel digestion.

Q: How much protein coverage does a typical identification yield?
A: Coverage is very much dependent on the amount of protein present and also the nature of the protein itself. For abundant proteins coverage can be high as 70-80% whereas low abundance proteins can be identified from as little as one or two peptides (with sequence information).

Q: How much material do I need for the protein analysis?
A: Although the sensitivity of our mass spectrometers is very high, it is always prudent to send as much protein as possible. We can identify (or obtain sequence) from anything that can be seen by coomassie or SyproÔ Ruby protein stain. Silver is more problematic due to the nature of the stain and its interaction with the protein but we obtain signal from over 90% of samples. Listed below are some arbitrary numbers for you to get started.

Theoretical required total starting protein amounts for individualprotein visualization by staining
Protein abundance, copies per cell Silver staining* Coomassie staining*
Protein amount, mg# Number of cells Protein amount, mg# Number of cells
10 20.073 1.20x109 2007.3 1.20x1011
100 2.007 1.20x108 200.7 1.20x1010
1,000 0.201 1.20x107 20.1 1.20x109
10,000 0.020 1.20x106 2.0 1.20x108
100,000 0.002 1.20x105 0.2 1.20x107

* Protein detection limits for silver and Coomassie staining were 1 and 100ng respectively.
# Soluble yeast protein was calculated based on 1 mg of yeast protein being derived from harvesting 6.107; cells. Calculations are based on a protein molecular mass of 50 kDa and 100% efficiencies of the procedures used.

*Gygi, SP, Corthals, GL, et al (2000), PNAS, 97(17), 9394

 

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