Version 25.5.07
All operations, at least before stopping digestion, are preferably done in a clean dust free environment (air clean workstation, clean room etc.).
1. Cut the gel band of interest. Do not cut a sharp thin pieces. The width of the gel piece should be ~1 mm or more.
Note: There is a common notion of cutting a gel piece as close to its staining borders, which is not helpful or even wrong. The resolving power of all common gels is not high. Proteins usually migrate as rather wide bands. Gel staining however, frequently reveals a “tip of an iceberg”, and not the real spacial distribution of a protein in the gel.
When dealing with entire gel lane, slice a gel lane onto ~10-40 1-, 2- or 3-mm pieces with a Mickle gel slicer or any suitable cutter. On a copy of a gel picture mark the beginning, the end, and some intermediate positions of the slicer blade for future cross-reference and a gel slicing calibration. Use tweezers with a thin flat tips to pick up the pieces and to chop them onto ~1×1x1 mm pieces. All manipulation with gel pieces can be easily performed using a clean white or black plate. After slicing and dicing the gel pieces, transfer them directly into the 1.6 ml tubes, which were labeled and pre-filled beforehand with a ~1.5 ml of a distaining solution. For example, for coomassie stain use 1/1 v/v of 50mM ammonium bicarbonate and MeOH.
2. Distain for necessary period of time in ~1.5 ml distain solution with agitation. For example, for 10-20 min with Zn-distain, if gel was stained with Zn-stain, or for 30min-3 hours in coommasie distain solution (freshly made 50 mM ammonim bicarbonate in 50% MeOH, if the gel was stained with a colloidal coommasie. All reagents are the highest purity available). After distaining, remove solution as completely as possible.
Optional: Reduce and alkylate the proteins in the gel if needed at this point. Wash well the gel pieces after completion of the alkylation reaction.
3. Dehydrate the bands well. For this add 1ml of acetonitrile to all gel pieces and slightly agitate the vial for 10-15 min. All gel pieces should become very white color. Remove acetonitrile.
4. Dry the gel pieces in by leaving the tubes open. Optional: microwave for 1 min and let it dry.
Note: When the gel pieces become very dry, they have tendency to pop out of the tubes under the influence of static electricity that might be generated on the tubes after drying.
5. Dissolve well an aliquot of trypsin (2.5 ug) in 100 -200 uL of 50mM ammonium in a freshly made and filtered (0.45um filter) bicarbonate buffer, pH ~8. Add 5-10 ul of trypsin solution to gel pieces depending on an estimated volume of initial gel piece. Add just enough of trypsin solution to keep a gel piece slightly “thirsty” upon rehydration. Put the tubes on ice for 30-45 min ( to prevent self digestion of trypsin while gel rehydration)
6. After incubation on ice is finished, add ~10-20 ul of the same digestion buffer, but without trypsin.
7. Digest for 3-12 hours at 37 C.
8. Stop digestion and extract peptides by addition of 30-40 uL 7% formic acid/ 0.1% TFA in water. Also add ~1-2 ul of Porous 50 beads from a stock kept in methanol (beads/MeOH = ~1/5, v/v). The hydrophobic beads will act like a pump absorbing all peptides that diffuse out of the gel pieces.
Note: Amount of beads added should later create ~100-400 nL volume of bead column in the tip of a Gelloader tip or a Zip tip.
Gently shake the pieces of gel in the presence of beads for 3-12 hours in a medium speed shaker. For longer extraction times, ~ 4-12 h, should be done at 4C, to minimize hydrolysis of -D-P- bonds.
9. After extraction step, collect the beads in the bigger gel loader pipette trying to avoid any small gel pieces. By doing so, press the tip against the bottom of the vial and shake from side to side. While collecting aspirate several times to elute any beads that could stack to the pieces of gel or the walls of the vial. Transfer the collected beds into the Gelloader pipette the tip with a frit made from C8 membrane (3M). Collect the beads by gentle pushing the liquid and create a C18 column at the end of the tip. When processing many gel pieces use a centriguge designed to accommodate 30 GelLoader pipette columns on the flat rotor.
10. Wash the column with 5-20 uL of 0.1% TFA.
11. Elute with matrix solution of DHB dissolved in 60% methanol, 1-2% acetic acid.
If using 4hcca as a matrix, elute peptides with a solution with high content of organic solvent (60-70%+0.1% TFA or 2% acetic acid) directly to the target and then add the 4hcca matrix.
12. In case of using 4hcca as matrix, wash the final spot 2 times with 5-7 ul drops of 10% MeOH/0.1% TFA solution. Leave a droplet for 10-30 sec and remove the liquid by suction from a vacuum line.