Information for Submitting Samples
Sample Preparation:
NB. Keratin contamination will always be observed so extra precautions must be taken to minimize the amount of contamination!
1. Wear gloves at all times during sample preparation.
2. Thoroughly wash anything that will come into contact with your sample (i.e. gel apparatus, staining trays, gel excising implement and gel storage and shipping tubes).
3. After the gel has been run, non-specific dust contamination can still be introduced so keep samples covered or protected as much as possible.
Gel suggestions:
1. Use standard SDS-PAGE gels. Maximize the amount of protein on as minimal amount of gel as possible. A concentrated gel band works a lot better than a large amount on a large diffuse gel band. Run multiple lanes if necessary to obtain sufficient amounts of protein for analysis.
2. Gel thickness of 1mm is preferred. Gradient, denaturing and native gels are acceptable.
3. Stain with Coomassie Blue using standard conditions. Stain only until your bands are visible.
4. Destain gels thoroughly. A clear background is preferred. All excess stain must be removed otherwise residual stain artifacts may interfere with data acquisition.
5. Excise band as precise as possible. If there is any diffuse stained edges to the band, omit it and excise only the clearly stained band. Excise a similar size background slice and follow the same work up protocol.
6. Place gel slice into a washed, plain 1.5 ml Eppendorf tube. Do not use any tubes with o-rings.
7. Wash the gel slice with 50% HPLC Grade Acetonitrile/Water at least 2 times for 10 min with occasional vortex mixing. Discard the wash solution. Use plastic pipette tips to remove the solution otherwise the gel slice tends to stick to glass Pasteur pipets. This step helps wash out any residual detergent or salt.
8. Rinse the Eppendorf cap off with the 50% solution and close the cap tightly. No additional solution is needed to cover the gel band. Do not parafilm the tube. The gel band will remain moist and can be stored in a -20oC freezer or delivered to us.
