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.
- Thoroughly wash anything that will come into contact with your sample (i.e. centrifugal tubes, gel apparatus, staining trays, gel excising implements, gel storage equipment, etc) to remove keratins and other contaminants.
- Keratin contamination is a problem limiting protein identification. 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.
- Vials: rinsing of vials with 50% HPLC grade acetonitrile/water is recommended. For liquid samples, use the smallest vial necessary, (as low as to the 0.5-0.6ml vials). For gel samples, use a 1.5 ml tube. Do not use colored vials and vials with rubber o-rings or gasket. To minimize sample loss, use Eppendorf LoBind tubes (cat. # 22431081 or 22431064).
- Avoid using molecular mass cutoff filters (e.g. Centricon filters). If they must be used, thoroughly wash them before use.
- 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. If higher sensitivity is required, use Sypro Ruby staining. Sypro stains require a UV source to visualize gel spots or bands. Destain gels and 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. Do not mince up a gel bend into very small pieces. Use fresh (not used) staining or destaining solution.
Excise a band from a blank area of gel, about the same size as your sample gel bands, and label it 'gel blank.' You will not be charged for blank sample.
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.