Mar 28, 2025
Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) Cofractionation and In-Gel Digestion
- Harper JW1,
- Miguel A. Gonzalez-Lozano2
- 1harvard university;
- 2Harvard Medical School
Protocol Citation: Harper JW, Miguel A. Gonzalez-Lozano 2025. Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) Cofractionation and In-Gel Digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbzm2ygpk/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: October 18, 2024
Last Modified: March 28, 2025
Protocol Integer ID: 110309
Keywords: ASAPCRN, Blue-native page
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 000282
aligning science across parkinson's
Grant ID: 024268
Abstract
This protocol is used to isolate protein complexes by blue-native page, a type of native gel. The source of proteins for separation in this example is early endosomes isolated by Endo-IP (with Flag-tagged EEA1 as an affinity handle).
Materials
n-Dodecyl β-D-maltoside (DDM, Gold Biotechnologies, DDM5)
NativeMark Protein Standard (Invitrogen, LC0725)
NativePAGE 4-6% Gels (Invitrogen, BN1002BOX)
tris(2-carboxyethyl)phosphine (TCEP; Gold Biotechnology, 51805-45-9)
2-chloroacetamide (Sigma-Aldrich, C0267)
trypsin (Promega, V511C)
Lys-C (Wako Chemicals, 129-02541)
KPBS buffer (25 mM KCl, 100 mM potassium phosphate, 150 mM NaCl, pH 7.2)
MultiScreen Filter Plates (Sigma Millipore, MSHVN4510)
Sample Preparation
Sample Preparation
Generate the sample for analysis, such as a purified organelle (Endo-IP in this case). The relevant protocol for Endo-IP can be found at: https://doi.org/10.17504/protocols.io.ewov14pjyvr2/v2
Separation on BN-PAGE
Separation on BN-PAGE
Resuspend freshly prepared purified endosomal pellets in 40 μL of KPBS containing 0.5% n-Dodecyl β-D-maltoside (DDM).
Extract proteins by rotating the mixture for 45 minutes at 4°C.
Clarify the protein extracts by centrifugation at 20,000 x g for 20 minutes at 4°C.
Mix the clarified extracts with 10 μL of BN loading buffer, 1 μL of Coomassie G-250 mix, and 0.5 μL of a native molecular weight marker.
Load the samples onto a 4–16% NativePAGE gel.
Run the gel at 150 V for 1.5 hours, followed by 250 V for 20 minutes at 4°C.
Fix the gel in 50% ethanol and 3% phosphoric acid. Reconstitute in water.
Stain the fixed gel with Coomassie G-250 dye.
In-gel digestion
In-gel digestion
Slice each sample lane from the gel into 48 1-mm slices.
Transfer the gel slices to a 96-well filter plate for in-gel digestion.
Reduce the proteins by adding 100 μL of 5 mM tris(2-carboxyethyl)phosphine (TCEP) in 50 mM ammonium bicarbonate and incubate for 30 minutes at 37°C.
Alkylate the proteins with 20 mM chloroacetamide in 50 mM ammonium bicarbonate for 15 minutes at room temperature.
Destain with 50% acetonitrile in 50 mM ammonium bicarbonate, and dry in 100% acetonitrile.
Digest proteins with 0.2 μg Lys-C for 4 hours at 37°C, followed by an overnight incubation with 0.2 μg trypsin.
Extract the resulting peptides from the gel by increasing concentration of acetonitrile up to 70%.
Dry the peptide extracts using a SpeedVac concentrator. Reconstitute the dried peptides in a solution containing 5% acetonitrile and 5% formic acid.
Samples are now ready for liquid chromatography-tandem mass spectrometry (LC-MS/MS).