Jun 26, 2026
Detection of phosphorylated proteins
- Glenda Alquicer1
- 1Revmatologický ústav Oddělení experimentální revmatologie (ODER)
- Biochemistry - protein analysis
Protocol Citation: Glenda Alquicer 2026. Detection of phosphorylated proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxjr6kl8j/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: May 25, 2026
Last Modified: June 26, 2026
Protocol Integer ID: 317880
Keywords: western blot, detection on phospho proteins, phosphorylated proteins, protein detection, stripping, re-probing, phosphorylated proteins detection, proteins detection, phosphorylated protein, membrane, detection
Funders Acknowledgements:
Lubomira Papikova
Glenda Alquicer
Abstract
High-Sensitivity Detection of Phosphorylated Proteins Using a Single Membrane with Stripping and Re-probing
Guidelines
PBMC collection from rheumatoid arthritis patients for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee.
Materials
Peripheral blood mononuclear cell (PBMC) lysates
**Transfer membrane**
- VerdiBlot membrane (0.3 µm pore size)
- PVDF membrane brand A (0.2 µm pore size)
**Blocking Buffer**
- EveryBlot™ Blocking Buffer (Bio-Rad, Cat. No. 12010020)
**Antibodies**
- Phospho-STAT3 (Tyr705), clone D3A7 (Cat. #9145)
- STAT3, clone 79D7 (Cat. #4904)
- Phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), Cat. #9101
- p44/42 MAPK (ERK1/2), Cat. #4695
- anti-rabbit IgG HRP-linked, Cat. #7074
**Chemiluminescence**
- Clarity western ECL substrate (BioRad)
**Imaging System**
- Azure™ c300 Imaging System
**Software**
- ImageLab™ software (Bio-Rad)
- Microsoft Excel
**Stripping Buffer**
- 0.1 M glycine, pH 2.2 (HCl adjusted)
- 0.1% SDS
- 1% Tween-20
Protocol materials
EveryBlot™ Blocking Buffer (Bio-Rad, Cat. No. 12010020)BioRadCatalog #1201002
VerdiBlot 0.3 µm (LAM-X)VerdiBlot
Clarity Western ECL SubstrateBio-Rad LaboratoriesCatalog #1705060
Sample Preparation
Sample from rheumatoid arthritis patients were prepared and quantified for total protein content. Equal protein amounts were loaded (1, 10, 25, and 50 µg) onto 10% SDS-PAGE gels for separation under denaturing conditions.
Wet Transfer Procedure
2h
Following electrophoresis, gels and membranes were equilibrated in transfer buffer (Tris 25mM and 200mM Glycine). Transfer membrane used - VerdiBlot 0.3 µm (LAM-X)VerdiBlot . Filter paper and fiber pads were also soaked in transfer buffer prior to assembly. Transfers were performed in a cold room at constant voltage: 02:00:00
2h
Membrane Blocking and Antibody Incubations
Membranes were blocked with EveryBlot™ Blocking Buffer (Bio-Rad, Cat. No. 12010020)BioRadCatalog #1201002 .
Primary antibody incubation: 1:1000 dilution in blocking buffer, overnight at 4 °C with gentle shaking.
Secondary antibody incubation: HRP-conjugated antibody, 1:1000 dilution in blocking buffer, 1 hour at room temperature with shaking.
Protein Detection
Developed using chemiluminescence (Clarity Western ECL SubstrateBio-Rad LaboratoriesCatalog #1705060 ) and imaged on the Azure™ c300 Imaging System.
Colorimetric image for protein marker; chemiluminescence for blots (1min exposure).
Images analyzed using ImageLab™ software (Bio-Rad) and Microsoft Excel.
Reprobing by Mild Stripping
For total protein detection after phospho-protein analysis membranes required a mild glycine-based stripping protocol before reprobing.
Subsequently the membranes were washed in TBST (2 ×5 min) then incubated it in stripping buffer for 10-15 min at RT with agitation. Afterwards, washed 3 ×10 min in TBST and neutralized in TBS (1 ×10 min). Followed by incubation in blocking buffer (20 min). Antibody removal was comfirmmed by secondary-only incubation and rapid development (repeat stripping is recommended if necessary) then reprobed with the antibody of interest.