Mar 19, 2025
Protein Sample Preparation from Acrylamide Gel for Mass Spectrometry
- Patricia Yuste-Checa1,
- Barbara Steigenberger2,
- Andreas Bracher1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany;
- 2Mass Spectrometry Core Facility, Max Planck Institute of Biochemistry, Martinsried, Germany
Protocol Citation: Patricia Yuste-Checa, Barbara Steigenberger, Andreas Bracher, F Ulrich Hartl 2025. Protein Sample Preparation from Acrylamide Gel for Mass Spectrometry. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq6r33lk5/v1
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: March 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124566
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to prepare protein samples from polyacrylamide gels for mass spectrometry proteomics.
Materials
Digestion Buffer:
| A | B | |
| Tris-HCl | 25 mM | |
| Acetonitrile | 10% | |
| Trypsin | 10 ng µl–1 |
Protein Sample Preparation from Acrylamide Gel
Protein Sample Preparation from Acrylamide Gel
Slice the protein band of interest from a Coomassie blue-stained polyacrylamide gel using a scalpel.
Note
Avoid contamination by human keratin by using clean apparel and reagents. Wear gloves.
Wash thoroughly the gel pieces with 150 µL of de-staining buffer (25 millimolar (mM) ammonium bicarbonate, 50% ethanol).
Dehydrate the gel pieces with 150 µL of pure ethanol.
Remove the ethanol and dry the gel pieces using a SpeedVac.
Add 50 µL of digestion buffer (25 mM Tris-HCl, 10% acetonitrile, 10 ng µl–1 trypsin).
Digestion Buffer:
| A | B | |
| Tris-HCl | 25 mM | |
| Acetonitrile | 10% | |
| Trypsin | 10 ng µl–1 |
Cool On ice for 00:20:00 .
20m
Add 50 µL of 25 millimolar (mM) ammonium bicarbonate buffer.
Incubate Overnight at 37 °C .
8h
Collect the peptides released to the supernatant.
Extract additional peptides by incubation at 25 °C for 00:20:00 in 100 µL of extraction buffer (3% trifluoroacetic acid (TFA), 30% acetonitrile).
20m
Centrifuge at 4000 rpm, 00:02:00 .
2m
Collect the supernatant. Repeat steps 10-12 twice.
Dehydrate the gel pieces with 100 µL of pure acetonitrile at 25 °C during 00:10:00 .
10m
Centrifuge at 4000 rpm, 00:02:00 .
2m
Collect the supernatant and combine with previous supernatants.
Remove the acetonitrile using a SpeedVac.
Add 70 µL of 2 Molarity (M) Tris-HCl pH 8.0, 10 millimolar (mM) TCEP, and 40 millimolar (mM) chloroacetamide (CAA).
Incubate 00:30:00 at 37 °C .
30m
Acidify the peptides to 1% TFA.
For the desalting of the peptides, prepare a SDB-XC stage tip by pushing 3 plugs of 0.8 mm diameter from C18/SDB-XC and push them into a 300 µl pipette tip.
Wash with 20 µL of methanol to complete dryness by spinning.
Wash with 20 µL of buffer B (0.1% Formic acid and 80% acetonitrile in water). Spin the tips but make sure that the tip is not dry. A small volume of buffer B should be visible on the top.
Equilibrate the tips 2 times with 200 µL of buffer A (0.1% formic acid in water).
Load the sample onto the stage tips and spin at 1500 x g .
Wash the tip with 100 µL of buffer A and spin to dryness.
Elute the peptides from the tip using 60 µL of buffer B.
After elution, speed-vac the samples at 30 °C for 00:40:00 and store at -20 °C until loading on the LC-MS system.
40m