Sample Preparation Considerations

Sample prep is one of the oft-overlooked steps in achieving a high quality proteomic experiment. Despite the enormous technical advances in mass spectrometry instrumentation and software, the simplest samples can easily overwhelm the most advanced mass spectrometer and bad samples can actually render a mass spectrometer unusable.

MAXIM – the cleaner your sample the better your data.

If you wish to have success do not give us samples that contain:

• significant particulate or insoluble material
• radioactive or biohazardous substances

Another significant challenge in mass spec enabled protein ID is contamination. Keratin is a common ubiquitous contaminant that can either be a minor irritant or a major impediment to successfully completing your experiment, and is often introduced in the handling of protein gels. These tips will improve your chances of success!

• Analytical grade reagents and MilliQ water should always be used.
• Wear a lab coat and nitrile gloves at all times when handling your samples. After putting on gloves, rinse them under a stream of clean water before handling gels.
• Store gels in meticulously clean, covered plastic containers. Use opaque containers for fluorescent stains or wrap with foil. If you wash gel storage and staining containers with detergent, a thorough rinse with methanol is recommended.
• We strongly recommend the use of pre-cast gels and reagents. We have had excellent success with Bio-Rad Criterion and Ready gels (Standard or XT chemistry).
• Do not re-use reagents at any time.

Please see the general comments regarding good laboratory practice in staining gels. The guidelines are generally applicable. We do not recommend using containers previously used for blotting, since they are often contaminated with blocking proteins.

We recommend:

• Eppendorf brand tubes (non-siliconized and non-colored).
• The use of volatile buffers such as H2O, Methanol, Acetonitrile, Ammonium Bicarbonate, Acetic acid, Formic acid, Trifluoroacetic acid.
• All reagents should be of the highest quality available.
• If your sample needs to be filtered please use Millipore Ultrafree-MC. Other filters can contaminate your sample.