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Practical Notes

Because samples and goals for experiment can vary considerably,it is important that we have as much information as possible before we begin an analysis. Important information and why we need it are listed below:

  • Information re. you and your lab. We need this information to set up an account for you.
  • Sample amount. Why? Depending on available amount we will be able to guide with respect to analysis strategy. We appreciate that often it is not possible to give exact amount numbers but even estimates can be helpful.  For example: barely visible by silver stain or heavy stained by coomassie blue and: ~1ug vs ~100ug.
  • Sample matrix.Why? Because this allows us to plan needed sample preparation ahead of time. For example, if your samples contain detergent, additional steps are required.
  • Sample name(s). Why? Because this helps to understand your experiment and discuss your data.
  • Taxonomy. Why? We want to search your data using the correct data base. For example, if your rat protein is expressed in human cells, it is appropriate to search both data bases which can allow us to find unexpected proteins. Searching ‘all species’ is often a very messy affair because of homology.
  • Any relevant protein sequence information.Why? Because sometimes you work with a mutated or a tagged form that is not in our data base. If your protein sequence is not in our data base, then we are ‘blind’ to your protein.
  • Goal/expectation for an analysis. Why? Because we always aim to tailor an analysis to answer the asked question.
  • Sample order. Why? For many sample sets it is not practical to randomize injections, and we prefer to analyze from lower to higher amounts to minimize potential carry-over. Also, if you expect a protein to only be present in ‘treatment’, we will want to analyze ‘control’ before ‘treatment’. You can find more examples here and you are always welcome to discuss with the PRC team.
  • Any expected or known outcomes. Why? Making us aware of any expected outcomes can be quite valuable to us. This can be especially useful when “QC-ing” the data analysis to understand if we are on the right track. For example, with “Co-IP” experiments, we like to know the name and species of the “bait” protein and any known interactors/binding-partners you may be aware of.

 

Comments related to different sample types

  • SDS-PAGE gel bands. Make sure that the samples are dust free. Though silver staining is sensitive to the eye we experience that same sample amount stained by Colloidal Blue performs significantly better when analyzed by LC-MS and we suggest using the latter. Colloidal blue is more sensitive than Coomassie blue. Should you decide to excise the gel band your self, be careful to avoid keratin contamination.  At all time, where gloves, cover the container where you store your gel and do not lean-in over the gel. Gels and gel bands can be stored in water at 4°C  for 2-3 days.  The PRC can help excise the gel bands – and for those cases it is helpful if a image of the gel is available.
  • Affinity Purification samples (aka co-ip’s). For magnetic beads, drop off the sample ‘dry’ (use the magnet to remove the last salt wash). A ‘dry’ sample can be stored at -20°C if drop-off is not possible right away. For agarose beads we prefer the sample in a pH 8 buffer such as TRIS, HEPES or 50 mM ammonium bicarbonate.  Samples stored in buffer should be dropped off to the Center ASAP. Always keep cold to minimize dissociation from the beads. For APEX and BioID type experiments the proteins of interest are strongly bound and we believe that quick drop-off is less urgent – refer to guidelines.
  • Proteomics profiling/analysis of cell lysates/tissue. Protein concentration measurements and/or some quality control is very useful. It can also be very helpful to talk to the scientist in the Center since tissue types can vary greatly. Some guidelines and thoughts are available.
  • Polar Metabolites. We prefer dry samples. Following our guideline is very important.
  • Lipid samples. We prefer dry samples. See SOP for guidelines.
  • DNA/RNA. We require that samples are digested to nucleosides and submitted dry. See guidelines.
  • MHC peptides. We do not enrich MHC peptides but we can analyze them by LC-MS/MS. Amount from MHC-peptide enrichment experiments are often very low and we suggest that samples are prepared in low protein binding tubes/vials, are desalted and cleaned-up using STAGE tips and that the cleaned-up peptides are eluted into sample vials compatible with our auto sampler (orange cap vials) – the latter avoids transfer step which can reduce losses.

 

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