May 27, 2026
Detection of GFP-tagged multispanning transmembrane proteins expressed in HEK293T cells
- Danara Vonk1,2,
- Celena Stuitje1,
- Mark S. Hipp1,3,
- Anja U. Bräuer2,4
- 1Department of Biomedical Sciences, University Groningen, University Medical Centre Groningen, Groningen, Netherlands;
- 2Department of Human Medicine, Division of Anatomy, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany;
- 3School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Oldenburg, Germany.;
- 4Research Center of Neurosensory Science, Carl von Ossietzky University Oldenburg, Oldenburg, Germany
Protocol Citation: Danara Vonk, Celena Stuitje, Mark S. Hipp, Anja U. Bräuer 2026. Detection of GFP-tagged multispanning transmembrane proteins expressed in HEK293T cells . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2dk13g1y/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: April 30, 2026
Last Modified: May 27, 2026
Protocol Integer ID: 316043
Keywords: membrane protein, multispanning, western blot, multispanning membrane protein, multispanning transmembrane protein, transmembrane protein, detection of gfp, membrane, gfp, overlapping band, western blot protocol
Abstract
Western Blot protocol optimized for the detection and analysis of multispanning membrane proteins specifically. Protocol provides improved resolution and separation of partially overlapping bands for multiple multispanning membrane proteins tagged with GFP.
Materials
DMEM (Gibco; 41966-029)
FBS (Pan Biotech; P30-3306)
10.000 Units/mL Penicillin/10.000 µg/mL streptomycin (Gibco; 15140-122)
CO2 incubator
Plasmid DNA
PEI (Polysciences; 23966)
Tris (Sigma Aldrich; 0000336028)
SDS (Bio-Rad; 1610418)
Triton X-100 (Sigma Aldrich; X100-160mL)
Glycerol (Sigma Aldrich; G5516-1L)
Bromophenol blue (Sigma Aldrich; B0126-25G)
Glycine (Sigma Aldrich; 33226-1KG)
Ponceau S (Sigma Aldrich; P3504)
Glacial Acetic Acid
Tween-20 (Sigma Aldrich; P1379)
ß-mercapthoethanol (Sigma Aldrich; M3148)
30% Acrylamide/0.8% Bisacrylamide (National Diagnostics; EC890)
TEMED (Sigma Aldrich; T9281)
APS (Sigma Aldrich; A3678-25G)
Mouse-anti-GFP antibody (JL-8) (Takara Bio Living Colours; 632381)
Goat-anti-mouse Alexa 680-conjugated antibody (Life Technologies; A-21058)
Cell scraper
Sonication device (Branson SFX-150)
Centrifuge
Heating block
Licor Odyssey Fc imaging system
Bio-Rad Mini-PROTEAN Tetra Cell
Bio-Rad Tetra Blotting Module
Nitrocellulose membrane (Bio-Rad; 1620112)
Skim-milk powder (Zuidmolen Groesbeek)
Troubleshooting
Problem
High signal-to-noise ratio
Solution
Try different temperatures for heat incubation after addition of Laemli buffer
Safety warnings
Protocol uses multiple toxic chemicals, make sure that you have checked all safety requirements for the chemicals used and work with protective equipment when required.
Cell Seeding
Seed 3 x 106 cells in 10 cm dishes in DMEM medium supplemented with 10% FBS and 100 units/mL penicillin and 100 µg/mL streptomycin.
Grow cells overnight at 37°C and 5% CO2.
Transfection
Tube A: mix 5 µg of plasmid DNA with 500 µL DMEM medium without additives.
Tube B: mix 10 µL PEI (1 mg/mL) with DMEM medium without additives.
Incubate both tubes for 5 minutes at room temperature.
Add the contents of Tube B to Tube A.
Incubate for 30 minutes at room temperature.
Gently add 800 µL of transfection mix to the cells in a dropwise manner.
Grow cells overnight at 37°C and 5% CO2.
Prepare Buffers
If storage temperature is not specifically indicated, store buffer at room temperature.
Lysis Buffer I
50 mM Tris pH 7.4
150 mM NaCl
1% v/v Triton X-100
0.1% v/v SDS
5 mM EDTA
Store at 4°C
Tris 1
0.5M Tris pH 6.8
0.1% v/v SDS
Tris 2
1.5M Tris pH 8.8
0.1% v/v SDS
Laemli Buffer (5x)
12.5% w/v SDS
50% v/v Glycerol
300 mM Tris pH 6.8
0.05% w/v Bromophenol Blue
20% v/v ß-mercapthoethanol
Store at -20°C
Running Buffer (10x)
0.25M Tris
1.92 M Glycine
1% w/v SDS
Transfer Buffer (1.25x)
25 mM Tris
192 mM Glycine
Store at 4°C
Ponceau S staining solution
0.1% w/v Ponceau S
5% v/v Glacial Acetic Acid
PBS-T
1x PBS
0.1% v/v Tween-20
Prepare SDS-PAGE Gels
Separation Gel
Table 1 - Gel recipe separation gel SDS-PAGE
Stacking Gel
Table 2 - Gel recipe stacking gel SDS-PAGE
Cell Lysis
Wash cells with 1x PBS.
Add 500 µL Lysis Buffer to one 10 cm dish and scrape cells off directly afterwards.
Collect scraped material in a 1.5 mL tube.
Sonicate samples for 3 x 3 seconds at 50% amplitude (Settings optimized for Branson sonication devices).
Incubate samples on ice for 30 minutes.
Centrifuge at 10.000 x g for 10 minutes at 4°C.
Transfer supernatant to new tube.
SDS-PAGE
Determine total protein concentration of samples.
Prepare samples containing an end concentration of 2 µg/µL protein and 1 x Laemli buffer.
Heat samples for 5 minutes at 55°C.
Load samples on desired percentage SDS-PAGE gel and run at 100V for 10 minutes.
Increase voltage to 150V and let proteins migrate through the SDS-PAGE gel as far as possible before proteins start running of the gel.
Western Blot
Prepare 1x Transfer Buffer by adding 200 mL 100% methanol to 800 mL 1.25x Transfer Buffer.
Blot proteins on a nitrocellulose membrane at 110V for 70 minutes at 4°C by wet-blotting.
Prepare a 10% skim-milk in PBS-T blocking solution.
Incubate membrane in Ponceau S staining solution for 3 minutes on a shaker to check for complete transfer.
Wash membrane 3x 5 min with PBS-T on a shaker.
Incubate membrane for 1 hour in blocking solution at room temperature on a shaker.
Quickly wash membrane 2-3x in PBS-T to remove blocking solution completely.
Incubate membrane in mouse-anti GFP antibody (Takara Bio Living Colours, JL-8; 1:1000) in 3% BSA in PBST overnight at 4°C on a roller.
Wash membrane 3x 5 minutes in PBS-T on a shaker.
Incubate membrane in goat-anti-mouse Alexa 680-conjugated antibody (Life Technologies; 1:7500) in 3% BSA in PBST for 1.5 hours at room temperature and protect from light.
Wash membrane 3x 5 minutes in PBS-T on a shaker.
Develop protein signal on a Licor Odyssey Fc imaging system at 700 nm.