Scripps Research Logo

Center for Protein and Nucleic Acid Research

In-Gel Digest Protocol

  1. Load the samples as concentrated as possible on a normal SDS-PAGE (at least 50 pMoles). Please load 50 pMoles of BSA, HSA, a-lac, or some known protein in one lane.
  2. Rinse your gel with water.
  3. Stain the gel with Coomassie for 1-1.5 hr (1 g of Coomassie G-250, 400 mL MeOH, 100 mL Acetic Acid, 500 mL water)
  4. Destain until the bands are resolved using 30% MeOH/water.
  5. Excise bands of interest, the control bands, and a blank gel piece with a scalpel rinsed in ethanol and put the bands in an eppendorf tube. The blank gel piece should be as large as the band of interest. Once in the tubes, cut the bands into 3-5 smaller pieces.
  6. Add enough 0.2 M ammonium bicarbonate:50% acetonitrile to cover the gel pieces. Incubate for 30-60 minutes at 30oC.
  7. Discard the liquid and repeat the wash.
  8. Dry the pieces completely under a slow stream of nitrogen for approximately 20 minutes.
  9. Rehydrate the gel pieces by adding 2-5 uL of 0.2 M ammonium bicarbonate.
  10. Immediately add 0.5 ug of modified sequence grade trypsin from Promega dissolved in the buffer given by Promega at a concentration of 0.1 mg/ mL. A control digest should also be run of at least 50 pMoles of a known protein in 100 uL 0.2M ammonium bicarbonate.
  11. Add repeated additions of 2-5 uL of 0.2 M ammonium bicarbonate until the bands are covered in buffer.
  12. Incubate with agitation at 30oC overnight.
  13. The Control Digest Should Be Run On the Hplc Before Proceeding.
  14. Transfer the supernatant liquid to a new eppendorf.
  15. Extract the fragments from the band using 100-500 mL of 60% CH3CN/ 0.01% TFA (enough to cover the pieces). Agitate for 20 minutes at 30oC. Repeat.
  16. Combine with the supernatant liquid from step 14.
  17. Dry to approximately 150 uL.
  18. Run on a microprobe RP-HPLC using a 0.5% gradient from 0.1% TFA/water to 60%, 0.08% TFA in CH3CN on a C18 column while collecting the peaks.

This procedure was adapted from "Improvement of an "In-Gel" Digestion Procedure for the Micropreparation of Internal Protein Fragments for Amino Acid Sequencing.", Helmann, U., Wernstedt, C., Gonez, J., Heldin, C., Anal. Biochem. 224, 451-455 (1995).