May 15, 2025
Version 2
BAF_Protocol_001a In-Gel Digestion V.2
- 1University of Virginia
- Nicholas Sherman: Biomolecular Analysis Facility Core
Protocol Citation: Nicholas Sherman 2025. BAF_Protocol_001a In-Gel Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn7r5mv5d/v2Version created by Nicholas Sherman
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 21, 2022
Last Modified: May 15, 2025
Protocol Integer ID: 217744
Keywords: proteomics, mass spectrometry, digestion
Abstract
This protocol is for in-gel digestion of proteins, including large mixtures, to produce peptides for mass spectrometry analysis. The gel method can be useful for getting rid of detergents or small molecules that might interfere with minimal loss. The input is a gel band up to 1cm x 1cm (do not use more gel as gel background rises faster than peptide signal - instead cut into sections). The output is a relatively clean peptide digest that is ready for quick cleanup by C18 tip.
Guidelines
1. Wash hands before starting. Refrain from wearing heavy lotions or perfumes.
2. Do not wear clothing that contains wool or a lot of loose fibers.
3. Use nitrile gloves (not latex).
4. Perform all procedures under PCR clean hood cabinet.
5. All sample preparation must use polypropylene tubes cleaned by addition/removal of EtOH (pure ethyl alcohol), then H2O, then EtOH. Dry in clean hood for several hours upside down. This strips off polymer coatings.
7. All reagents must be prepared in small glass bottles (polypropylene lined caps - no glue foil).
8. Solutions should be prepared fresh.
Materials
Pre-cleaned microtubes 1.5 mL - USA scientific, SEAL-RITE® 1.5 mL MICROCENTRIFUGE TUBES color: natural, part number:1615-5500.
Pre-cleaned microtubes 0.5 mL - USA Scientific, SEAL-RITE® 0.5 mL MICROCENTRIFUGE TUBES color: natural, part number: 1605-0020.
Screw cap microtubes 1.5 mL - Fisher Brand, Conical Screw Cap Tube, color: natural, part number: 02-681-339.
20mL liquid scintillation vials PE cone lined cap - Wheaton DWK986756
ABC - Fluka analytical, Ammonium Bicarbonate, Sigma Aldrich, part number: 09830.
DTT - Fisher BioReagents, Dithiothreitol, part number: BP 172-5.
IA - Sigma Aldrich, Iodoacetamide, part number: 1149.
Trypsin: Promega, Sequencing grade modified, it comes frozen (20 µg in 40µL of 50 mM Acetic Acid), part number: V511C.
FA - Fisher Chemical, Formic Acid, Optima™ LC/MS Grade, part number: A117-50.
MeOH - Fisher Chemical, Methanol, Optima™ LC/MS Grade, part number: A456-4.
EtOH - Fisher Chemical, Ethyl Alcohol/Ethanol, 99.5%, Millipore Sigma, part number: MEX02763.
ACN - Fisher Chemical, Acetonitrile, Optima™ LC/MS Grade, part number: A955-4.
Acetic Acid - Sigma-Aldrich ≥ 99.99%, part number: 338826-100 ML.
H2O - Fisher Chemical, Water, Optima™ LC/MS Grade, part number: W6-4.
Syringe - Unimetrics PKS250, 250µL Peek Laboratory Syringe
Pipette tips 10 and 200 µL, CAT: 05-408-186, CAT: 02-681-151 - Fisher Brand.
Pipette tip 1000 µL, CAT: 1111-2721 - USA Scientific.
Gel-Loading tips, 1-200 µL, CAT: 02-707-81- Fisher Brand.
2 to 20 µL Micropipette - Gilson™ F144056MT
10 to 100 C Micropipette - Gilson™ F144057MT
20 to 200 µL Micropipette - Gilson™ F144058MT
100 to 1000 µL Micropipette - Gilson™ F144059MT
Tweezer and scalpel
White light Transilluminator, 115V, 60Hz, 1.2 Amps, part number: 95-0214-01.
Incubator chamber or Eppendorf thermomixer for temperature control.
Before start
REAGENTS: (All reagents to be prepared fresh for each digestion)
1. EtOH 75 % ethanol in distilled H2O.
2. Destain solution: 10 mL MeOH, 10 mL H2O, 500 μL Acetic Acid.
3. 100 mM ABC, MW: 79.08 g/M: 0.158 g in 20 mL distilled H2O.
4. 50 mM ABC: 0.079g in 20 mL distilled H2O.
5. 10mM DTT, MW: 154.253 g/M: 0.0015 g in 1 mL of 100 mM ABC (Use screw caps microtubes. DO NOT mix until directly before you are ready to use)
6. 50 mM IA, MW: 184.962 g/M: 0.0092 g in 1 mL of 100 mM ABC (Use screw caps microtubes. DO NOT mix until directly before you are ready to use)
7. Trypsin: Take out the enzyme from -20C and keep it on ice just before two steps to be use.
8. Extraction solution 1: 5% FA in H2O. 10 mL volume (Solution can be use for a couple weeks)
9. Extraction solution 2: 5% FA in 50% ACN/H2O. 10 mL volume
*Buffers may keep at RT for up to two weeks but reagents such as DTT, IA and trypsin must be fresh right before use.
DAY 1:
DAY 1:
19h 30m
19h 30m
Clean tweezer and scalpel with EtOH 75%. Spread EtOH on the transilluminator area you will use to cut the bands and clean it up by sliding the kimwipes in only one direction.
10m
Cut gel band with the scalpel into small pieces (~2 mm) on the transilluminator.
Transfer the pieces with the scalpel/tweezer in a pre-cleaned 1.5 mL polypropylene tube.
5m
Add 500 μL of destain solution to the pieces. Let rest for ~2hrs at RT (Room Temperature).
2h
Discard destain (using 1 gel load tip for all tubes in batch) and replace with another 500 μL of destain solution. Let rest for 30min. It’s okay if some stain remains. It will be removed during further wash steps.
30m
Discard the destain solution and dehydrate gel slices by adding 200 μL ACN. Let rest for 5 min.
5m
Discard ACN and repeat step 6 dehydration.
5m
Reduce the proteins by adding 30 μL of 10 mM DTT. Incubate for 30 min at RT.
30m
Discard the DTT solution.
1m
Alkylate the proteins by adding 30 μL of 50 mM IA. Incubate it for 30 min at RT, in dark. (get ice and keep the 50 mM ABC solution on it).
30m
Discard the IA solution.
1m
Wash gel pieces with 100 μL of 100 mM ABC for 10 min and discard the solution.
10m
Dehydrate gel pieces in 200 μL ACN for approximately 5 min and discard the ACN.
5m
Rehydrate gel pieces in 200 μL 100 mM ABC for 10 min.
10m
Discard ABC solution.
1m
Dehydrate gel slices in 200 μL ACN approximately 5 min. Discard ACN and repeat this step. (Take out the Trypsin from -20C and keep it on ice).
5m
Prepare trypsin at (20μg/mL) by adding 960 μL cold 50 mM ABC.
2m
Add 30-50 μL of the trypsin solution to cover the gel pieces and rehydrate on ice for 30 min.
30m
Microfuge and remove any excess trypsin solution and add 5-20 μL of 50 mM ABC. Incubate the reaction overnight at 37°C.
16h
Day 2:
Day 2:
3h 40m
3h 40m
Prepare Extraction Solutions:
a. Extraction Solution 1: H2O/Formic Acid: 9.5 mL H2O/0.5 mL FA
b. Extraction Solution 2: ACN/H2O/Formic Acid: 5 mL ACN/4.5 mL H2O/0.5 mL FA
10m
DO NOT remove excess ABC supernatant from yesterday!!!
Add 10 μL Extraction Solution 1 to each tube (or more if needed to cover the gel pieces). Let rest for 10 minutes. Transfer the supernatant to a pre-cleaned 0.5 mL tube.
10m
Add 10 μL Extraction Solution 2. Let rest for 10 min. Transfer the supernatant to the same 0.5 mL tube.
10m
Repeat the 10 µL Extraction Solution procedure (steps 22-23). Transfer the supernatant to the same 0.5 mL tube.
10m
Evaporate the sample via speed vac and reconstitute to 10-100 μL total volume (depending on the amount of total protein) with 0.1% Formic acid. Most gel bands contain amount of protein that can be resuspended in 20 μL.
2h
Perform desalting using C18 tips (BAF_Protocol_003):
– 10 µL tips for a small amount of proteins
– 100 µL for a higher amount of proteins.
1h