Jun 15, 2026
Trapping S-glutathionylated proteins by a biotinylated glutathione-ethyl-ester probe-mediated enrichment method from living cells: A modified biotin-switch technique.
- Ajanta Chatterji1,2,
- ajanta Chatterji3
- 1Program in Cellular and Molecular Medicine, Faculty of Medicine, The Ottawa Hospital Research Institute, University of Ottawa, Ottawa, ON, Canada.;
- 2Presently, the author has moved to the University of Oxford, Oxford, United Kingdom.;
- 3The Ottawa Hospital research Institute
- Ajanta
Protocol Citation: Ajanta Chatterji, ajanta Chatterji 2026. Trapping S-glutathionylated proteins by a biotinylated glutathione-ethyl-ester probe-mediated enrichment method from living cells: A modified biotin-switch technique.. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl83mrrv2w/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: June 13, 2026
Last Modified: June 15, 2026
Protocol Integer ID: 319104
Keywords: modified biotin switch assay, biotin switch assay, glutathionylated protein, modification of protein, biogee assay, glutathionylation, biotinylated glutathione, modified biotin, cellulo detection of this modification, existing assay, other similar assay, assay, proteins in living cell, protein, identified cysteine, increased oxidative stress condition, such as thiol alkylation, dependent protein, thiol alkylation, ester probe, cellulo detection, mediated enrichment method, antibody, oxidative stress condition
Abstract
Detection of S-glutathionylation is inherently challenging because of its reversible nature, which makes it difficult to identify proteins undergoing this oxidative post-translational modification (PTM). This prompted researchers to identify methods suitable to trap this modification, making it imperative to study this PTM in a metabolically active state, such as from living cells. There have been several approaches that are used for identifying this modification of proteins taking place in a test tube; however, limited success and false positive signals have been observed for in-cellulo detection of this modification. Several advancements have been made to the existing methods to circumvent the cumbersome steps and the limitations of the existing assays. In contrast to the most traditional approaches, such as thiol alkylation and direct detection approaches, or antibody-based detection approaches, and other techniques, this BioGEE assay is a modified biotin switch assay previously reported by Sylvian et al., (2002) [16], which is further modified and updated, presenting several advantages and is readily available as a kit. However, no single protocol exists as a collated and comprehensive guide for experimentalists exclusively designed for this assay and requires modifications and optimization from existing protocols from the literature that are designed for other similar assays to detect different PTMS. Herein, we report an up-to-date, modified, comprehensive, reliable, and handy protocol that uses a membrane-permeant biotinylated glutathione-ethyl ester (BioGEE) probe to identify S-glutathionylated proteins in living cells, with which we have recently identified cysteine-dependent proteins undergoing this often reversible modification, S-glutathionylation, under increased oxidative stress condition.
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Materials
**Buffers**
(i) RIPA lysis buffer: 50 mM Tris-HCl, 150mM NaCl, 0.1% (w/v) sodium dodecyl sulphate (SDS), 0.5% (w/v) sodium deoxycholate, 1% Triton X-100.
(ii) 10X Phosphate buffer saline (PBS)- 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, and 18 mM KH2PO4. Adjust PH- 7.4.
(iii) PBS wash solutions: (a) 1X PBS, (b) 1X PBS + 50mM Iodoacetamide (IAA), 1X PBS+ 0.1% SDS.
(iv) 3mg/ml D-Biotin solution: 3mg/ml D-biotin was weighed and dissolved in alkaline water (PH 10.3-10.5)
(v) SDS PAGE buffers: (a) Sample loading dye buffer (10% SDS, 250 mM Tris HCl (PH 6.8), 50% glycerol, 0.5% bromophenol blue)
(b) SDS PAGE Tank running buffer 1X MES- 50mM MES, 50mM Tris, 0.1% SDS, and 1mM EDTA
(vi) Western blotting buffers- (i) Wet Transfer Buffer (Tris base, Glycine and NaCl)
(ii) Blocking solution (either 5% skimmed milk or 5% BSA)
(iii) Washing buffers (PBST (Phosphate buffer saline + Tween 20)- 100 mL of 10X PBS stock solution, 1 mL of Tween-20, and 900 mL of distilled water
**Reagents and Chemicals**
(i) BioGEE Probe Kit was obtained from Thermo Fisher Scientific. (CAT# G36000)
(ii) Streptavidin magnetic beads (Thermo Fisher Scientific CAT# 88817)- Beads were stored at 4°C upon receiving. The manufacturer’s protocol was followed, and the beads were mixed thoroughly and equilibrated by pre-washing the beads 3x with sample lysis buffer or PBS before the experiment.
(iii) DTT (Dithiothreitol) was obtained from (Thermo Fisher Scientific, CAT#R0861)- DTT is always freshly prepared either for elution by dissolving 10mM DTT in PBS + 0.1% SDS wash solution buffer or for adding in sample loading dye (100mM DTT) for breaking the disulfide bond of proteins in the sample during SDS-PAGE.
(iv) IAA (Iodoacetamide) was obtained from Thermo Fisher Scientific (CAT# 122270050) - 50mM IAA solution is prepared using PBS.
(v) Protease inhibitor cocktail (Thermo Fisher Scientific CAT#A32965)
(vi) Millipore Amicon 30kDA cut-off filter with collection tubes (Sigma Aldrich CAT #UFC901024)
(vii) BCA Assay protein estimation kit was obtained from (Thermo Fisher Scientific, CAT#A55864)
(viii) NuPAGE pre-casted gels 4-12% bis-Tris gradient gels, 10 wells (Invitrogen CAT# WG1401BOX)
(ix) Primary antibody (for this experiment, we have used rabbit polyclonal anti-Parkin antibody Cell Signalling, CAT# 2132)
(x) Secondary antibody (goat anti-Rabbit IgG antibody CAT# A32731)
(xi) Chemiluminescence kit: ECL developing solutions (Thermo Scientific CAT# 34580) .
(xii) All other apparatus used and chemicals to prepare buffer solutions were purchased from Thermo Fisher Scientific and Sigma-Aldrich.
BioGEE Assay Method on cultured living cells
The experiment can either be paused at this point or continued the same day by proceeding with the subsequent lysis steps. To pause, snap freeze the sample (which is incubated in the lysis buffer step 7) and store in -80 °C freezer.
To the supernatant, add 200 µL of ice-cold 1X RIPA lysis buffer containing 50mM IAA + 1X protease inhibitor cocktail; incubate on ice for 30 min.
If samples have been snap frozen, then on the day of the experiment, lysates must be thawed on ice and then proceed with the lysis steps. Sonicate the samples at 20% amplitude for 10 seconds, two rounds, and then centrifuge at maximum speed for 10 minutes at 4 °C.
Estimate protein concentration in the sample using any standard protein estimation assay, such as the BCA method.
Keep 20-40 µL of lysate aside for the input lane on immunoblotting.
Streptavidin-Biotin pull-down of BioGEE tagged proteins (Day-2)
Biotin-Streptavidin pre-blocking step must be performed freshly on the same day of the experiment. Prepare biotin-blocked streptavidin beads by adding 500 µL of 3 mg/mL D-biotin to 50 µL of streptavidin magnetic beads (10:1 ratio) and incubate with rocking for 1 hour at RT. Wash 5 times with PBS using a magnetic rack.
Add biotin-blocked streptavidin beads from above to the sample (~50 µL) and incubate for 30 min with rocking in a cold room or 4 °C. This step is essential as it prevents any non-specific binding from cell lysate, such as cells’ endogenous biotin or free biotin, from binding to streptavidin beads and ensures only BioGEE-labeled proteins get bound to streptavidin beads. Pellet the magnetic beads.
Now, carefully take the supernatant and transfer it to 50 µl of freshly equilibrated streptavidin-magnetic beads (to equilibrate, pre-wash the streptavidin beads 3 times with PBS) and incubate for 2 hours with rocking at 4 °C.
Pellet beads using a magnetic rack and transfer the supernatant in the tube labeled as “pre-clear” or “unbound” and store at -20 °C.
The beads from above would now contain BioGEE-tagged proteins from the sample. Wash the beads 3 times with 10X volume of ice-cold RIPA.
Then wash the beads 2 times with 10x Volume of 0.1% SDS in PBS. Save the supernatant and label it as “wash” in a new tube. Store in – 20 °C freezer for future, if needed to run on immunoblot.
Resuspend magnetic beads-bound sample proteins in 1 volume of 0.1% SDS in PBS and incubate for 30 min at RT with rocking. This step allows the removal of any non-specific proteins that were bound to the beads but were not labelled with the BioGEE probe, such as endogenous biotin-containing proteins or enzymes.
Pellet beads. Transfer the supernatant to a new tube and label as “–DTT eluate”. This would serve as a negative control in the western blot while analyzing BioGEE tagged proteins. Store at – 20 °C.
Resuspend beads in 1 volume of 0.1 % SDS + 10 mM DTT in PBS and incubate for 30 min at RT with rocking. This step allows the removal of proteins tagged with BioGEE and bound to the beads as DTT cleaves the disulfide bond formed between the BioGEE and reactive cysteine residues of proteins, thereby releasing the proteins in the supernatant. Pellet beads.
Transfer supernatant to a new tube and label as “+DTT eluate”. This supernatant would contain the proteins that underwent S-glutathionylation with the BioGEE labelling.
Transfer samples to Molecular weight cut-off filters (such as 10 or 30kDA) of appropriate size depending upon the molecular weight of the protein of interest and concentrate by centrifugation at 14 000 x g for 20 min at 4 °C. The retentate was transferred into collection tubes and centrifuged at 1000 x g for 5 min at 4 °C to collect the concentrate.
Freshly weigh 100mM DTT and add to the 1X sample loading dye buffer, which was then mixed with the samples, making an equal concentration of proteins to be loaded in SDS PAGE (adjust volumes with PBS if needed) and store all the prepared tubes in loading dye buffer (+ DTT eluate, -DTT eluate, input control) store at – 20 °C.
Immunoblotting (Days 3-4)
Thaw samples on ice. Heat the samples on a heat block at 95 °C for 5-10 mins. Resolve the samples by running in SDS PAGE (4-12 % gradient gel electrophoresis).
Perform immunoblotting by transferring the proteins to PVDF membrane (activated by 100% methanol), followed by blocking (in blocking solution), and incubating with the primary antibody of the protein of interest (at appropriate dilution). After washing with PBS wash solutions and secondary antibody incubation (at appropriate dilution), bands were observed by chemiluminescence.
Protocol references
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Acknowledgements
I am grateful to Dr. Michael Schlossmacher (MGS) for letting me use his laboratory space and resources to conduct experiments and publish original data in the past using this protocol. I am thankful to the funding agencies, Canadian Institute of Health Research (CIHR), for funding the parkin project awarded to MGOs and Parkinson’s Research Consortium of Ottawa (2022-2024), Shelby Hayter fellowship awarded to me (AC) for conducting the research work using this protocol. I am thankful to MGS lab colleagues who helped me with scientific discussions and technical assistance during work with the method in the past. I am very grateful to Dr. Rajib Sengupta for his tireless motivation, valuable discussions, and mentorship in the past. I am also grateful to Prof. Dr. Arne Holmgren (deceased), a Redox Biology pioneer and member of the Nobel Society, Karolinska Institute, for his invaluable suggestions and guidance/mentoring of our team members in the past.
**Funding: This work was supported by a Parkinson’s Research Consortium of Ottawa fellowship awarded to AC (Shelby Hayter award 2022-2024).
**Availability of Data and Materials: Data supporting the results, materials, and resources associated with this protocol can be found in previously published [17], and in the main text, figures, and supplementary figures of this manuscript. All other supporting information is available online in publicly archived databases such as PubMed. The protocol is also available on protocols.io. [link]
**Conflict of Interest: There is no potential conflict of interest to declare.