Protein Sample Preparation Guide
The mailing kit contains detailed instructions on sample handling and shipping. Prior to sending non-gel-based samples, please complete the sample submission form. This lets us know what to expect and when, so we can start on your samples immediately.
Please ship samples via FedEx Priority Overnight service to:
14153 Robert Paris Ct.
Chantilly, VA 20151
Frozen samples should always be shipped on 3-5 kg. of dry ice.
Please, do not ship frozen samples on Fridays.
Gel spots or bands and FFPE samples may be shipped dry or suspended in water at ambient temperature. Prior to shipping, store at 4 C.
Sample Preparation Considerations
Optimal protein sample preparation is a prerequisite to optimal results.
In general, these two rules can help to ensure high quality results:
- Less sample preparation is better.
- More steps in the process often leads to more opportunities for error and consequently for innacurate or biased results.
- Let us do the sample preparation.
- We have optimized our methods to yield the best possible results while minimizing bias
Tips for handling specific protein sample types.
Gel spots or bands: can become contaminated with other proteins, mainly keratins, by contact or by exposure to dust. To minimize contamination use nitrile gloves to handle gels and keep all equipment which contacts the gel clean by rinsing with HPLC grade water (gel plates, scalpels, punches, staining trays, and the interior of the tube you will put the gel into!). Use pre-cast gels. Stain using vendor kits. We recommend CBB, but if silver staining, use a mass spectrometry-compatible silver stain kit from one of the major vendors. Gel bands and spots may be shipped at ambient temperature. If desired, add sufficient water or 0.1% acetic acid to cover gel.
Wrap tube lids with ParaFilm if you have any doubt about the ability of your tube to remain closed during shipping or use screw-cap tubes. Be aware that ParaFilm can introduce contaminants that can lower overall assay sensitivity.
Snap freeze samples using liquid nitrogen or a dry ice/ethanol bath. Be sure to freeze your samples in an upright position to keep the sample away from the lid/cap.
Ship frozen. Large volumes (> 10 mL) should be precipitated by acetone precipitation or a method of your choice. Ship frozen.
Pellet in an isotonic buffer such as PBS to prevent lysis. Wash the pelleted cells several times with the same buffer to remove media components. Ship frozen.
Can become contaminated with cells released by the needle or scalpel used to access the fluid. These can be removed by using a centrifugal filtration device such as Millipore Ultrafree or Pierce Spin Cup. Ship frozen.
Cell culture supernatants
Large volumes (> 10 mL) should be precipitated by acetone precipitation or a method of your choice.
Phosphorylation analysis samples
Lyse the cells or disrupt the tissue in a buffer consisting of 8 M urea, 20 mM HEPES pH 7.0, 75 mM beta-glycerophosphate, 1 mM sodium vanadate, 1 mM DTT, 1.5 mM EGTA. Ship frozen.
Fresh frozen tissue
Avoid use of optimal cutting temperature (OCT) medium if possible. Ship frozen.
Ship as-is, at ambient temperature.
Protocols for Shotgun Proteomics Sample Preparation
We preform these methods in-house using optimized protocols. The content below is provided for informational purposes.
Sequencing grade trypsin from Promega is widely used. Their in-gel digestion protocol is here.
Filter-Assisted Sample Preparation (FASP)
Popularized by the Mann Lab, this method permits protein extraction with SDS and heat, which will generally extract more proteins than other mehods, by removing the SDS with a molecular weight cut-off filter device. Digestion is performed in the device and recently the group revised the method to incorporate multi-enzyme digestion and fractionation. More details are available at their site.
Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
Developed by the Mann Lab, this method permits quantitative comparison of two or more cell cultures, one of which is grown in the presence of stable isotope labeled amino acids. The resulting proteins, digested to peptides, have a mass offset equal to the isotope label mass, enabling comparison to the unlabeled sample by inspection of the peptide peak intensities. The classic protocol is here.
Titanium dioxide-based enrichment has largely replaced IMAC or other metal-based enrichment methodologies as the method of choice. It was originally described here.
Protocol from Pierce.