- 2D-gel :
- SDS-PAGE :
50 fmol of your protein (if known) or 40ug of total protein amount.
- Bands/spots :
silver staining exposition time cannot exceed 5 min
- IP : 50 fmol of the protein of interest
- Protein and peptide in solution : 50 fmol.
If your samples have detergents and surfactants (SDS, Tween, NP40, Triton...) as low as 0.01%, they will have to be run on SDS-PAGE. Surfactants and detergents deteriorate HPLC columns and alter mass spectrometer signal.
You can cut the bands yourself and put them in an eppendorf tube without liquid. Please note that combining many bands in the same tube might make peptide digestion/extraction less efficient.
If you cannot elute your protein from the antibody, you can run your sample on a SDS-PAGE gel because a large amount of digested antibody will hide the MS signal of your digested protein.
The enzyme used for digestion is trypsin. Trypsin cleaves at the C-terminal ends of lysine and arginine. If you want a digestion with another enzyme you will have to provide it.
Phosphopeptide enrichment (large scale)
Minimal amount of proteins should be 250 ug. It is your responsibility to perform the cell lysate and to measure the protein concentration by Bradford or micro-BCA.
- If the sample is a 1D-band, silver staining can't be used since it might induce sulfation and be mistaken with phosphorylation: http://www.ncbi.nlm.nih.gov/pubmed/18936056)
- Our ability to identify a protein phosphorylation site depends on the sample purity. High abundance of contaminating proteins might block low-abundance phosphopeptide identification
- Mass spectrometer mass range allows the identification of peptides from 7 to 25 amino-acids. The shorter the phosphopeptide, the higher the identification confidence is. To maximize identification probability, another digestion enzyme than trypsin might be needed.
If your protein is not wild type, you will have to provide us its complete sequence and not mutation positions only.
Samples must be submitted
between 9am and 5pm to:
Eric Bonneil / Chantal Durette / Francis McManus
IRIC-Pavillon Marcelle Coutu
2950 chemin Polytechnique
Montreal, QC, H3T 1J4
T: (514)-343-6111 ext. 0646
You will also have to fill the service request form, on which you will have to specify the NAME of your PI and the sample taxonomy.
Protein/peptide abundance can be compared across replicates or conditions. However, abundances for the same protein can only be compared across samples and not abundances of different proteins altogether.
Delays : 2 weeks.
Price : see table
Billing :The bill will be sent directly to your PI. No PO is required.