Proteins separated by gel electrophoresis
Many of the samples we deal with are proteins separated by gel electrophoresis. Gel bands following 1D, or spots following 2D gel electrophoresis must be cut before submission.
Please keep in mind the following considerations!!!
Beware of contaminations in all phases of sample handling. Dust particles contain high levels of proteinaceous contaminations (hair and skin particles, fuzzy chlothes) therefore carry out all staining steps in a closed dish, use chemicals and solutions dedicated to "mass spectrometry". Wash all equipments very thoroughly, filter solutions if necessary. Wear (power free) gloves and lab coats at each step, cut the bands/spots under a laminar flow hood and do not touch the vials at any stage of the sample preparation without gloves.
There are several methods applied to visualize proteins after gel electrophoresis. Unfortunately not all of them are compatible with the mass spectrometric analysis. Coomassie staining techniques are in general ideal for these investigations. However, there are some cases, when higher sensitivity is needed. Note, that silver staining applying glutaraldehyde in the senzitizing solution is not compatible with mass spectrometry! For this reason we recommend to use Coomassie staining, or an alternative silver staining protocol (.rtf). In case of silver staining do not overstain the gel, keep the background clear! According to our experiences, overstaining reduces the yield of identifiable peptides from the sample substantially!
Keep the volume of the empty gel matrix as small as possible, cut away unstained gel materials.
Submit the samples as soon as possible, freeze them only, if you can guarantee, that they do not thaw during trasfer to our lab. If you send the sample via mail, please contact us before posting it and send it on dry ice.
Protein solutions or pellets
Please note, that samples should not contain high amounts of salts (> 100 mM), non volatile buffer components (e.g. phosphate buffer) and ionic detergents (e.g. SDS, Triton). Keep in mind, that during protein purification, or lyophilisation cheap plastic ware or low quality solvents stored in plastic cans can contaminate your sample with plasticizers (polymeric material), which can be retained during chromatographic purification and reduce the yield of peptides, or make the analysis completely impossible.
Protein solutions should be frozen and delivered to us on ice. If you send the sample via mail, please contact us before posting it and send it on dry ice.
©ECs February 2007