In-gel tryptic digestion of Coomassie-stained or SYPRO-stained protein bands and sample preparation for LC-MS analysis
Protein bands of interest are excised, the resulting gel slices are cleaned, and Cys residues in proteins are in-gel reduced and alkylated. The gel slices are cleaned again, dehydrated with acetonitrile, rehydrated with trypsin solution, and allowed to incubate for several hours at 37 oC. Tryptic peptides are extracted from gel slices and prepared for MS analysis.
SDS or native PAGE
Run, stain, and destain your gel following your usual protocol. Use deionized water for the final destaining step.
Cut out only the intensely stained portion of each band for better results.
Materials and equipment
Ammonium bicarbonate buffers should not be stored longer than 1 month at 4 oC. Prepare reducing and alkylating solutions fresh. Store IAA in the dark at 4 oC protected from humidity.
- Acetonitrile (ACN)
- 2 mg/mL ammonium bicarbonate in 50% ACN (destaining buffer)
- 2 mg/mL ammonium bicarbonate in 9% ACN (digestion buffer)
- 2 mg/mL ammonium bicarbonate (overlaying buffer)
- Reducing reagent, either (a) or (b) in 2 mg/mL ammonium bicarbonate: (a) 15 mg/mL dithiothreitol (DTT); (b) 30 mg/mL Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)
- Alkylating reagent, 18 mg/mL iodoacetamide (IAA) in 2 mg/mL ammonium bicarbonate
- Proteomics grade trypsin (e.g. Sigma T6567-5x20UG, 5 vials x 20 ug lyophilized powder; or Promega V5113, 5 vials of frozen liquid, 40 uL of 0.5 ug/uL trypsin solution; or similar)
- 1 mM HCl for trypsin stock solution (to dissolve lyophilized trypsin)
- Dry block or other temperature-controlled incubator at 37-40 oC
- 5% formic acid in 50% ACN (peptide extraction solution)
- Access to a SpeedVac
- 0.1% formic acid in 3% ACN (peptide sample solution, optional)
- Access to a centrifuge capable of achieving 10,000 x g (optional)
Preparation of trypsin for digest
One vial (20 ug) of trypsin is sufficient for 100 1mm x 5mm x 1mm gel slices or 30-50 wider slices. One gel slice can be rehydrated with 6-20 uL trypsin working solution (more for large slices). Working solution must be prepared fresh and kept on ice throughout the rehydration procedure (step 7).
- Lyophilized trypsin
0.2 ug/uL stock solution: Reconstitute 20 ug lyophilized trypsin in 100 uL 1 mM HCl, mix gently to ensure complete dissolution, do not vortex. This stock can be aliquoted and stored for up to 4 weeks at -20 oC and will be stable for up to 3 freeze-thaw cycles.
20 ng/uL working solution: Dilute acidic trypsin stock solution 1/10 with ice-cold digestion buffer.
- Promega trypsin solution
0.5 ug/uL stock solution: Thaw a vial in a refrigerator or on ice. This stock can be aliquoted and stored at -20 oC.
12.5 ng/uL working solution: Dilute the 0.5 ug/uL stock solution 1/40 with ice-cold overlaying buffer, mix gently, do not vortex.
Volumes are given for 1 gel slice; increase volumes to completely cover multiple gel slices.
- Wash each gel slice in 200 uL of destaining buffer for 30 min with occasional vortexing, discard destaining buffer, repeat.
- Add enough reducing reagent to completely cover the gel slices (~ 50 uL per gel slice), vortex and spin down the slices in a bench-top centrifuge. (a) DTT: incubate at 60-80 oC 10 min (e.g. in a hot water bath); (b) TCEP: incubate at room temperature 10 min. Discard reducing reagent.
- Add enough alkylating reagent to completely cover the gel slices, vortex, spin down, and incubate 30-45 min at room temperature in the dark. Discard alkylating reagent.
- Wash each gel slice in 200 uL of destaining buffer for 15 min with occasional vortexing, discard destaining buffer, repeat.
- At this point, large gel slices should be diced into 1-2 cu. mm pieces for more efficient rehydration in step 7. Keep in mind that very small pieces can be accidentally drawn into a pipette tip and discarded.
- Dehydrate gel slices by incubating them in 50 - 100 uL ACN 2 – 3 times until the gel is completely white and rigid. Discard ACN and leave gel slices to dry in open tubes at 37 oC for 10-15 min. (Time from start 3.5 - 4 hrs. This could be a stopping point. Dehydrated gel slices should be stored in a freezer until next step.)
- Add 6-10 uL trypsin working solution directly onto the gel pieces and allow them to rehydrate on ice, repeating if necessary, until gel is completely transparent.
- Discard any trypsin solution that is not absorbed by the gel.
- Overlay the gel slices with the overlaying buffer to prevent drying out during the digestion (80 – 100 uL). Incubate at 37 – 39 oC for at least 3 hours or overnight.
- Transfer solution from step 9 into a clean micro-centrifuge tube. Cover the gel slices with peptide extraction solution, vortex for 20-30 min, sonicate for 15 min, spin down, and combine the resulting extraction solution with the solution from step 9.
- Repeat the extraction as described in step 10.
- Evaporate the combined solutions to dryness in a SpeedVac.
LC-MS sample preparation (optional)
It is best to submit the dried samples, because it may be necessary to store them for several days prior to analysis.
- Use only the HPLC-grade solvents and mass spec grade formic acid! Dissolve the dried peptides in 10-20 uL of 3% ACN containing 0.1% formic acid, sonicate for 2-5 min, vortex 2-5 min.
- Centrifuge for 15 min at 10,000 x g to remove particulates and carefully pipette out the supernatant without disturbing the bottom 2-3 uL of liquid and transfer it into an autosampler vial.
Tips for better results
- Keratin contamination
Main sources of keratin in the samples are skin, hair, and dust.
To minimize keratin contamination, wear clean gloves throughout the entire process of protein isolation and purification and don’t touch your hair, skin, or clothes with gloved hands.
Keep all gel equipment, supplies, and solutions protected from dust.
Use a clean razor blade for cutting out protein bands; rinse and wipe it before cutting another band.
- Positive controls
Run a protein standard in one lane as a positive control, cut out the band, and process it in the same way as you do your protein spots or bands of interest. If your sample analysis doesn’t work as expected, we can analyze your positive control and our standard to identify the problem.