This post is concerned to the In-gel digestion protocol introduce the digestion of a protein present in a SDS-PAGE gel band with trypsin, which can be extracted from either a 1D or 2D electrophoresis gel.
Reagents:preparing all reagents immediately prior to use. The water used in all components of the procedure is 17 megaohm/cm Nanopure or MilliQ-type water. The acetonitrile and methanol are HPLC grade. All other reagents should be the highest possible commercial grade available. As for the trypsin used here, it is a kind of sequencing-grade enzyme, which has been modified to inhibit autolysis and to minimize non-tryptic protease activities. Putting all reagents into 20-mL glass tube with Teflon lined caps or 1.5-mL plastic microcentrifuge tubes.
Mixing 10mL of methanol with 5 mL of water and 1 mL of acetic acid and then adjusting the total volume to 20 mL with water. The final concentrations should be 50% methanol and 5%acetic acid.
2. 100 mM ammonium bicarbonate: dissolving 0.2 g of ammonium bicarbonate in 20 mL of water.
3. 50 mM ammonium bicarbonate: mixing 2 mL of water with 2 mL of the 100 mL ammonium bicarbonate.
4. 10 mM DTT: placing 1.5 mg of dithiothreitol in a 1.5 mL plastic centrifuge tube, adding 1.0 mL of 100 mM ammonium bicarbonate and then dissolving the dithiothreitol.
5. 100 mM iodoacetamide: Placing 18 mg of iodoacetamide in a 1.5 mL plastic centrifuge tube. And then 1.0 mL of 100mM ammonium bicarbonate should be added and dissolving the iodoacetamide.
6.Trypsin solution: mixing 1.0 mL of ice cold 50 mM ammonium bicarbonate with 20 μg of sequencing-grade modified trypsin. Dissolving the trypsin through drawing the solution into and out of the pipette. The trypsin solution should be kept cold on the ice until use. The final concentration should be 20 ng/μL trypsin.
7.Extraction buffer: adding 10 mL of acetonitrile to 5 mL of water and 1 mL of formic acid and then the total volume should be adjusted to 20 mL with water. 50% acetonitrile and 5% formic acid should be the final concentration.
The 0.5 or 1.5 mL plastic microcentrifuge tubes used here are purchased as low-bingding, siliconized tubes. Rinsing all tubes in USP-grade ethanil and air-dried before use.
These are some steps before the digestion procedure, which should be introduced in our next post