Pierce cell surface protein isolation kit

Total and membrane protein extracts of HEK293 cells were obtained using the Pierce Cell Surface Protein Isolation Kit (89881; Thermo Fisher Scientific). Then, 40 µg of protein (total and membrane) was separated by electrophoresis on a 7% SDS–polyacrylamide gel and transferred to a PVDF membrane.
Blots were preincubated with 5% low-fat milk in TBS–0.1% Tween for 1 h at room temperature and incubated overnight at 4°C with β1-AR (ab3442) (Sun et al, 2021 (link)) or β2-AR (ab182136) (Cellini et al, 2021 (link)) antibodies from Abcam or E-cadherin antibody (3195) (Ye et al, 2022 (link)) from Cell Signaling Technology (as a positive control for membrane proteins) diluted 1:1,000 for each case. After washing of the primary antibodies, binding was visualized using a secondary horseradish peroxidase (Thermo Fisher Scientific)–labeled anti-rabbit antibody (for adrenergic receptors; 1:10,000 dilution) and anti-mouse antibody (for E-cadherin; 1:10,000 dilution) and enhanced with diaminobenzidine at 100 µg/ml in TBS with 30% H₂O₂.

The isolation of sperm surface proteins from non-capacitated, IVC, proteasomally inhibited IVC, and vehicle control IVC spermatozoa was done as we described earlier [53 (link)]. Briefly, a Thermo Scientific Pierce Cell Surface Protein Isolation kit was used according to the manufacturer’s protocol. In this method, mammalian cells were first labeled with EZ-Link Sulfo-NHS-SS-Biotin, which is a thiol-cleavable amine-reactive biotinylation reagent. Cells were subsequently lysed with a mild detergent, and labeled proteins were then isolated with NeutrAvidin^TM immobilized on agarose beads. The bound proteins were released via incubation with an SDS loading buffer (50 mM Tris∙HCl, pH 6.8, 1% (v/v) glycerol, 2% (w/v) SDS) containing 100 mM DTT. To achieve satisfactory protein yields, three column equivalents per treatment group were used for a single MS run. Three volumes of acetone were added to each extract and the samples were stored at −25 °C prior to the high-resolution mass spectrometry.

Five 100 mm Petri dishes with HEK293 cells expressing the different NMDAR constructs were washed three times with ice-cold PBS, pH 8.0, to remove any contaminating proteins.
Cells were suspended at a concentration of ~10 million cells/ml in PBS, pH 8.0. 1 mg of sulfo-NHS-LC-Biotin (Thermo Scientific) per ml of reaction volume was added to the cells and incubated at 4 °C for 30 min. After this, cells were washed three times with ice-cold PBS and 100 mM glycine to quench any remaining biotinylation reagent. The cell surface proteins are now biotinylated on exposed lysine residues.
Biotinylated proteins were purified using the Thermo Scientific Pierce Cell Surface Protein Isolation Kit^® following manufactured instructions. For loading control of western blots, an aliquot of total protein (before purification of biotinylated protein) was immunoprecipitated using anti-GFP selective polyclonal antibody (Thermo Scientific, A-11122), and the immunoprecipitation kit (Abcam ab206996) following the manufacturer instructions (both NMDAR subunits had GFP fused). For the identification of the NMDAR on the western blots, the selective rabbit monoclonal antibody (ab109182) was used for NR1 and antibody (ab133265) for NR2A (Abcam).

Membrane proteins were isolated using the Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Scientific, USA) as per the manufacturer’s instructions. Briefly, cells were biotinylated (EZ-Link Sulfo-NHS-SS-Biotin) in ice-cold PBS, quenched (ice-cold PBS+ buffer plus 0.1% glycine), and lysed. The lysate was passed through the Neutr-Avidin agarose columns. The columns were washed and bound proteins were eluted with the elution buffer.

Cell surface protein extraction and quantification were performed using, respectively, the Pierce Cell Surface Protein Isolation kit and the Pierce 660 nm Protein Assay (Thermo Scientific, Rockford, IL, USA), according to the manufacturer's instructions.
In order to obtain sufficient protein concentration for mass spectrometry analyses, human samples were combined in pools: MM-PC (n = 8) and ND-PC (n = 7).

Protein samples were prepared from cells using standard methods and were subjected to SDS-PAGE and transferred to a nitrocellulose membrane by wet western blot transfer (Bio-Rad) [12 (link)]. The membrane was blocked in TBST buffer (TBS + 1% Tween) containing 5% milk for 1 hour at room temperature. The blocked membranes were incubated overnight at 4°C with specific primary antibodies (Odyssey blocking buffer, 927–40000). The membranes were washed 3 times with TBST buffer, and then incubated with specific secondary antibodies provided by LI-COR for 2 hours at room temperature. After 3 washes with TBST buffer, the membranes were analyzed using the ODYSSEY Infrared Imaging system (LI-COR). Analysis of biotinylated cell surface proteins was performed using the Pierce Cell Surface Protein Isolation Kit (89881) from Thermo Scientific (Waltham, MA) [12 (link)]. Briefly, cells were labeled with Sulfo-NHS-SS-Biotin, a thiol-cleavable amine-reactive biotinylation reagent, and then lysed with mild detergent. Biotinylated surface proteins were isolated with Avidin Agarose, and eluted using SDS-PAGE sample buffer containing 50mM DTT. Samples were then analyzed for HER2 by immunoblot as above. All immunoblot experiments were performed at least 3 times and representative blots are shown in the figures.