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10 × Radioimmunoprecipitation assay buffer (RIPA)
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Protease and phosphatase inhibitors, including Phenylmethylsulfonyl fluoride (PMSF)
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Refrigerated centrifuge
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Microcentrifuge tubes
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BCA™ Protein Assay (Fisher Scientific, Hampton, NH, cat # PI23225)
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Betamercaptoethanol (BME, Sigma, St. Louis, MO, cat # M6250)
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Gel electrophoresis running equipment
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4–15% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad, Hercules, CA, cat # 4561084)
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10 × Running Buffer (Bio-Rad, cat # 161-0732)
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Distilled water
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trans-Blot Turbo Transfer System (Bio-Rad, cat # 17001917)
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- Tris-buffered saline containing 0.1% Tween 20 (TBST)
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10 × TBS (Fisher Scientific, cat # BP24711)
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Tween 20 (Sigma, cat # P9416)
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Ultrapure water
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Graduated cylinder
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Stir bar
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Glass bottle
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Stir plate
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- 5% nonfat milk in TBST
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1 × TBST
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Powdered nonfat milk
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Conical tube
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Rotator or shaker
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- 5% bovine serum albumin (BSA)
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1 × TBST
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BSA (Fisher Scientific, cat # BP9706100)
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Conical tube
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Rotator or shaker
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- Antibodies for exosome markers
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CD81 (Abnova, Taipei, Taiwan, cat # MAB6435)
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CD9 (Abcam, Cambridge, United Kingdom, cat # ab2215)
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ALIX (Abcam, cat # ab186429)
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- Antibodies for non-exosome markers, such as embryonic stem cell markers
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Nanog (Cell Signaling, Danvers, MA, cat # 3580S)
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OCT-4 (Cell Signaling, cat # 2750S)
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Clarity Western ECL Substrate (Bio-Rad, cat # 1705061)
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ChemiDoc™ Imaging System (Bio-Rad, cat # 12003153)
5.2 Methods
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Add 5 μL protease and phosphatase inhibitors to 500 μL 10 × RIPA.
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Mix gently then pipet sufficient 10 × RIPA/inhibitors solution into exosome samples resuspended in 1 × PBS to make a 1 × RIPA/inhibitor solution.
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- Vortex for 30 s, sonicate for 1 min then let exosomes sit on ice for 30 min.
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If not using immediately, flash freeze exosomes in liquid nitrogen and store at − 80 °C. Thaw on ice prior to use.
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Centrifuge samples at 12,000 RPM for 15 min at 4 °C.
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Transfer supernatant to a clean microcentrifuge tube.
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Quantify total protein using the BCA™ Protein Assay following manufacturer’s instructions
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Calculate the amount of protein to be run on the gel.
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- Prepare sample for gel loading by combing protein, RIPA buffer, sample buffer and BME and heat samples at 100 °C for 5 min. Immediately transfer to ice.
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Note: Tetraspanin antibodies may require non-reducing conditions. Check antibody sheet for details.
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Prepare running buffer then assemble gel running apparatus.
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Rinse each well prior to loading molecular weight markers and samples.
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Run gel at 120–150 V for about 1 h.
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Remove gels from apparatus and transfer to PVDF membrane following manufacturer instructions.
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After transfer, rinse membrane briefly with TBST then transfer to 5% milk solution for at least 1 h while shaking gently.
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Prepare antibody solutions in either 5% milk or 5% BSA in TBST.
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Briefly rinse the membrane with TBST prior to adding antibody solution then incubate overnight at 4 °C while gently rocking.
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Wash membrane three times with TBST for 10 min each while shaking.
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Add secondary antibody diluted in 5% milk and incubate for 1 h while gently shaking.
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Wash membrane three times with TBST for 10 min each while shaking.
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Prepare the ECL substrate following manufacturer instructions.
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Apply enough prepared ECL substrate to cover entire blot.
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Image blots following manufacturer instructions. See Fig. 2C for representative western blot for exosome markers.
Western blot analyses
Cell pellets or midbrain tissue fragments were dissected under microscope control, and homogenized in 150 μL of a radioimmunoprecipitation assay buffer (RIPA), consisting of 50 mM Tris-HCI (pH 7.4), 1% NP40, 0.5% sodium-deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM sodium chloride and 2 mM EDTA. Protein concentrations were measured using the bicinchoninic acid (BCA) method, following manufacturer’s instructions (Thermo Fisher Scientific). Detection of LRRK2 was performed on membrane-enriched and cytosol fractions. Brain tissue from control or lesioned hemisphere, or control and silenced SHSY5Y cells were homogenized in five volumes of a buffer consisting of 10 mM HEPES-potassium hydroxide (pH 7.4), 0.25 M sucrose and proteinase inhibitor cocktail (Sigma, Saint Louis, MO, USA). Homogenates were centrifuged at 900 × g for 10 min.
Pellets were homogenized again and centrifuged at 900 × g for 10 min. The pellets were collected as nuclear fraction. Supernatants were centrifuged at 12,000 × g for 15 min obtaining a synaptosome-enriched pellet. Supernatants were further ultracentrifuged at 46,000 × g for 1 h; the obtained pellets were collected as membrane fraction, while supernatants were collected as cytosolic fraction. Proteins (30 μg) were denatured in SDS (8%) and β-mercaptoethanol (5%) at 95 °C for 5 min.
After separation by SDS-PAGE on a 4–15% gel (for NAPE-PLD), or on a 3–8% Tris Acetate gel (for LRRK2) under denaturing conditions, the proteins were electrotransferred to nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in tris-buffered saline (TBS) and incubated overnight with primary antibodies (anti-LRRK2 antibody and anti-NAPE-PLD antibody, Abcam, 1:5000 and 1:200, respectively) and anti-GAPDH antibody (1:2000; Abcam) in 1% milk-TBS containing 0.1% Tween-20, followed by incubation with horseradish peroxidase-linked anti-rabbit IgG antibody (1:5000, Millipore, Darmstadt, Germany) in TBS containing 0.1% Tween-20 at room temperature for 1 h. Finally, proteins were visualized using an electrochemical luminescence (ECL) kit (Bio-Rad, Hercules, CA, USA) and the images were obtained using a LAS-4000 lumino-image analyzer system (Fujifilm, Tokyo, Japan).
Biotinylation of cell surface proteins
Biotin is a small water soluble molecule, which can bind to many proteins at primary amines without alteration of their biological functions. Since biotin is charged, it cannot go through the cell membrane, and only the proteins with primary amines exposed on the cell membrane can be covalently labeled with biotin. The biotin tag can be used to purify biotin-labeled proteins by avidin, which is the natural ligand for biotin (Fig. 13.2A). Also, it has been found that under regular culture conditions, cells have very low cell surface GRP78 expression. However, ER stress induced by thapsigargin (Tg) significantly promotes cell surface localization of GRP78 (Fig. 13.2B–D; Zhang et al., 2010).
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