Sample Preparation Basics
- Before preparing the samples, read the sample preparation guidelines and the sample requirements. Call if anything is not clear.
- Samples should not contain any undissolved, particular matter; it will clog and irreversibly damage expensive columns.
- Keratin contamination is a problem limiting protein identification, especially in conjuction with in-gel digestion. Keep samples covered or protected as much as possible and wear gloves at all times during sample preparation. Do not touch keratin sources (skin, hair, etc) with outer working surface of gloves. The use of freshly prepared reagents for SDS-PAGE and Coomassie staining will greatly improve outcomes.
- To minimize sample loss, use Fisher Low Retention tubes (cat. # 02-681-320) or Eppendorf LoBind tubes (cat. # 22431081 or 22431064).
- For in-gel digestion, maximize the protein/gel ratio in gel spots; a concentrated gel band works much better than a large diffused gel band. Gel thickness of 1mm is optimal (recommended). Stain gels with Coomassie Blue until your bands are visible. Excise protein bands as precisely as possible. If there is any diffuse stained edges to the band, omit it and excise only the clearly stained part of the band. Dice gel bands into 2x2 mm cubes, being careful not to smear the gel into a paste. Use fresh (not used) staining or destaining solution.
Gel bands should not get dry. Put an excised bend into a clean 1.5 mL Eppendorf tube and cap the tube tightly to keep the gel slice moist; gel bands can be kept for 4-5 days at 4°C or at least six month at -20°C.
- Vial label: each vial with a sample must have a label including your name, the sample name (unique but short), and the date. Please label both the side and the top of a vial.
- If sent by mail, samples should be sent frozen on dry ice; first, put the tubes with frozen samples in a plastic bag or a larger (50 mL, for example) plastic tube with padding making sure that the tubes will remain capped and the samples will not get lost during transportation. Note that it is dangerous and illegal to tape shut a container with dry ice during shipping.
- In-solution digestion, courtesy of Mr. Todd Markowski, University of Minnesota CMSP
- “Universal” sample preparation or Filter Assisted Sample Preparation (FASP)
- In-gel digestion, EMBL
- In-gel digestion, Nature protocols
- Stage tip (microscale cleanup), courtesy of Mr. Todd Markowski