Guidelines for plasma membrane protein detection by surface biotinylation

Abstract

Plasma membrane proteins are crucial for signal transduction, trafficking, and cell-cell interactions, all of which are vital for cell survival. These proteins, including G-protein coupled receptors, ion channels, transporters, and receptors, are key drug targets due to their central role in receiving and amplifying cellular signals. However, the isolation and purification of plasma membrane proteins pose significant challenges because of their integration with phospholipid bilayers and the small fraction of these proteins present in the plasma membrane. Biotinylation, in combination with streptavidin beads, provides an effective method for surface protein analysis by specifically labeling surface proteins without penetrating the cell membrane, enabling precise isolation and analysis with minimal contamination. In this study, we describe a 1-step method for analyzing plasma membrane proteins that can be routinely implemented in many laboratories.

INTRODUCTION

Plasma membrane proteins are pivotal in their roles in signal transduction, trafficking, and cell-cell interactions, all of which are important for cell survival (Cho and Stahelin, 2005, Kim et al., 2023, Stalder and Gershlick, 2020). Membrane proteins are important considering that almost one-third of all human proteins are membrane proteins (Wallin and von Heijne, 1998). Since they are the first to receive and amplify signals, cell membrane proteins, such as G-protein coupled receptors, ion channels, transporters, and receptors, have been targeted for drug use (Tautermann, 2014). Proteins synthesized in the endoplasmic reticulum are translocated to the membrane via various intracellular trafficking pathways. Analysis of membrane surface proteins could reveal defects in the intracellular trafficking of cells or consequences of genetic mutation in membrane protein genes (Bertrand and Frizzell, 2003, Conn et al., 2007, Velier et al., 1998). Various human diseases have been associated with defects in cell membranes. These misfolded proteins fail to reach plasma membrane, resulting in the failure of proper protein function (Kim et al., 2018, Marinko et al., 2019).

To date, many efforts have been made to rescue such misfolded proteins traffic to the plasma membrane (Gee et al., 2018, Gee et al., 2011, Jung et al., 2016, Oh et al., 2023). Isolation and purification of plasma membrane proteins remain challenging due to their association with phospholipid bilayers, carbohydrates, and extracellular matrices (Cournia et al., 2015). Additionally, while some membrane proteins are present in the plasma membrane, many exist within the trafficking pathways, such as the endoplasmic reticulum and Golgi apparatus (Banfield, 2011). As a result, analyzing total cell lysates often fails to provide accurate information about the proper localization of these proteins.

Biotin molecules are effective tools for surface protein analysis because they form covalent bonds with free amine groups on surface proteins but are impermeable to cell membranes (de Boer et al., 2003) (Figs. 1 and 2). Moreover, their low interference with native proteins allows for the isolation of intact proteins with high accuracy and minimal contamination. The use of streptavidin beads during precipitation further enables the study of various proteins in a single-step process (Fig. 3).

Fig. 1.

Labeling biotin to protein. Primary amines, such as lysine residues or the amino termini of proteins, are readily labeled with biotin by N-hydroxysulfosuccinimide (NHS) esters.

Fig. 2.

Types of NHS-biotin molecules. Sulfo-NHS-SS-Biotin, which is cleavable by strong reducing agent, water-soluble, and membrane-impermeable, and thus used for cell surface biotinylation, is shown in the red box.

Fig. 3.

Schematic illustration of the step-by-step procedure of cell surface biotinylation. For more detailed information on each step, please refer to the manuscript.

MAIN BODY

Step 1. Cell Preparation

• 1.Seed cultured cells in a 6-well plate or a 60-mm culture dish, depending on the scale of the experiment. If the cells tend to detach easily, precoat the plate or dish with poly-l/d-lysine to enhance cell adhesion.
• 2.The following day, transfect the cells with plasmids corresponding to the protein of interest. For optimal transfection efficiency, ensure that the cells are 60% to 80% confluent. Typically, the cytomegalovirus or cytomegalovirus immediate enhancer/β-actin promoter is used for transient gene expression. When using 6-well plates, a total of 0.5 to 4 μg of plasmids is appropriate. Transfection reagents, such as Lipofectamine 2000 (#11668019, ThermoFisher Scientific) or Polyethylenimine MAX (#24765, Polysciences), can be used according to the manufacturer’s instructions.
• 3.After transfection, incubate the cells for 24 to 72 hours to allow for adequate protein expression.
• 4.For analyzing native proteins for plasma membrane expression, only the cell seeding step is required.

Step 2. Biotin Labeling on the Cell Surface

• 1.Incubate the plate on ice for 10 minutes. This step stops further trafficking or endocytosis, ensuring that only proteins currently present on the cell membrane are labeled with biotin.
• 2.Wash the cells twice with ice-cold phosphate-buffered saline (PBS).
• 3.Add 0.1 to 0.5 mg/ml of sulfo-NHS-SS-biotin (#21331, ThermoFisher Scientific) in ice-cold PBS and incubate at 4°C for 30 minutes in the dark with gentle agitation. Sulfo-NHS-SS-biotin covalently binds to free amine groups exposed on the cell surface and is impermeable to the cell membrane. Because sulfo-NHS–based reagents are prone to hydrolysis, dissolve the sulfo-NHS-SS-biotin in PBS immediately before use, and ensure that the biotin solution is adequately protected from light.
• 4.Wash the cells twice with PBS, then incubate them with PBS containing 1% bovine serum albumin at 4°C for 10 minutes to quench any unbound biotin. A 50 mM glycine solution in PBS can also be used for quenching the reaction.
• 5.Wash the cells twice with PBS before harvesting to remove any remaining quenching buffer.

Step 3. Cell Harvest

• 1.Collect cells into a 1.5-ml tube with lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1% Triton X-100, and 1× protease inhibitor cocktail (#04693116001, Sigma-Aldrich). We routinely use 200 to 300 μl lysis buffer per one 6-well plate sample.
• 2.To break cell membranes and homogenize the sample, briefly sonicate the cells. Excessive sonication may generate heat and cause protein aggregation. Alternatively, placing the sample on an end-over-end rotator at 4°C for 1 hour can also achieve the desired effect. Centrifuge homogenized samples for 15 minutes at 15,000×g, 4°C.
• 3.Transfer the supernatant to a new 1.5-ml tube. To determine the cellular protein concentration, perform a Bradford assay by measuring the absorbance at 595 nm. Harvested lysates can be stored at −20°C for up to 1 month.

Step 4. Streptavidin-Biotin Incubation

• 1.Add 50 μl of 50% NeutrAvidin (#29204, ThermoFisher Scientific) or Streptavidin (#20353, ThermoFisher Scientific) resins to a 1.5-ml tube. Gently resuspend the streptavidin beads by inverting or vortexing the bead suspension. Make sure the beads are fully mixed before use.
• 2.Equilibrate the beads by incubating them with 1 ml of lysis buffer (composition provided in Step 3) on an end-over-end rotator at 4°C for 1 hour. Briefly centrifuge the tube on the tabletop centrifuge and aspirate the supernatant solution. Repeat the equilibration wash once more. After the final wash, use a gel-loading tip to carefully and completely remove any remaining lysis buffer.
• 3.Add 500 to 1,000 μg of cell lysate harvested in Step 3 to the beads. Then, add an appropriate amount of lysis buffer to bring the total volume to 200 μl per sample.
• 4.Incubate the lysate-streptavidin mixture on an end-over-end rotator at 4°C for 4 hours to overnight.
• 5.Use a tabletop centrifuge and aspirate the supernatant solution. Add 1 ml lysis buffer and wash on an end-over-end rotator at 4°C for 5 minutes. Repeat the wash twice more. After the final wash, use a gel-loading tip to carefully and completely remove the remaining lysis buffer.

Step 5. Elution, Visualization, and Quantification

• 1.Elute biotinylated proteins in 1× sample buffer containing 225 mM Tris-HCl (pH 8.45), 6% glycerol, 2% sodium dodecyl sulfate, and 65 mM dithiothreitol. Incubate at 37°C for 60 minutes. Because membrane proteins tend to aggregate at high temperatures, we do not recommend elution at 90°C, which is routinely performed for nonmembrane proteins.
• 2.Resolve the eluted sample by SDS-PAGE and carry out immunoblotting.
• 3.When immunoblotting, always perform positive and negative control experiments to confirm experiments were done correctly. For the positive control, we use Na⁺/K⁺-ATPase antibody (#sc-28800, Santa Cruz Biotechnology), as this protein is abundant in the plasma membrane. For the negative control, Aldolase A (#sc-390733, Santa Cruz Biotechnology), a cytosolic house–keeping protein, is recommended.
• 4.Acquire images of the immunoblot using a chemiluminescence or fluorescence imaging system. Use image analysis software (eg, ImageJ, Fiji, or other densitometry software) to quantify band intensities (Chua et al., 2024). Normalize the protein-of-interest signals to a loading control or the total protein loading to account for variations in sample loading and transfer efficiency.

Funding and Support

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korean government (RS-2024-00438709 to H.Y.G.).

Author Contributions

J.W.R., H.W.C., and H.Y.G. wrote the manuscript. All authors thoroughly evaluated and approved the manuscript.

Declaration of Competing Interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author Heon Yung Gee is an Associate Editor for Molecules and Cells and was not involved in the editorial review or the decision to publish this article.