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3 protocols using TRPA1

Total protein was isolated from approximately 50 mg of snap-frozen vagina, bladder, and colon using Cell Extraction Buffer (Invitrogen, Grand Island, NY) containing Halt protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA) and Na3VO4 (Sigma, St. Louis, MO). Protein concentrations were determined using a DC protein assay (ThermoFisher). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad, Hercules, CA), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio- Rad). The membranes were blocked for 1 hour at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4°C with antisera to CRF1 (1:500; Millipore, Billerica, MA), CRF2 (1:800; Millipore), TRPV1 (1:1000; Alomone Labs, Jerusalem, Israel), or TRPA1 (1:1000; Aviva Systems Biology, San Diego, CA) and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA) diluted in 5% milk in TBST. Membranes were then washed with TBST and incubated for 1 hour with antirabbit secondary antibody (1:10,000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).
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Total proteins were isolated from approximately 50 mg of snap-frozen DRG, vagina, and colon tissue samples using Cell Extraction Buffer (Invitrogen, Grand Island, NY) containing Halt protease and phosphatase inhibitors (ThermoFisher Scientific, Waltham, MA) and Na3VO4 (Sigma, St. Louis, MO). Protein concentrations were determined using a DC protein assay (ThermoFisher). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad, Hercules, CA), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio-Rad). The membranes were blocked for 1 hour at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4°C with antisera to CRF1 (1:500; Millipore, Billerica, MA), TRPV1 (1:1000; Alomone Labs, Jerusalem, Israel), or TRPA1 (1:1000; Aviva Systems Biology, San Diego, CA) and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA) diluted in 5% milk in TBST. Membranes were then washed with TBST and incubated for 1 hour with anti-rabbit secondary antibody (1:10,000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).
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Total proteins were isolated from approximately 50 mg of snap-frozen DRG, vagina, and colon tissue samples using cell extraction buffer (Invitrogen) containing Halt protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA) and Na3VO4 (Sigma). Protein concentrations were determined using a DC protein assay (Thermo Fisher). Samples were reduced by heating to 95°C for 5 minutes in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4% to 12% Bis-Tris gels; Bio-Rad, Hercules, CA), and transferred to a nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio-Rad). The membranes were blocked for 1 hour at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4°C with antisera to CRF1 (1:500; Millipore, Billerica, MA), TRPV1 (1:1000; Alomone Labs, Jerusalem, Israel), or TRPA1 (1:1000; Aviva Systems Biology, San Diego, CA) and GAPDH (1:2000; Cell Signaling Technology, Danvers, MA) diluted in 5% milk in TBST. Membranes were then washed with TBST and incubated for 1 hour with anti-rabbit secondary antibody (1:10,000; Cell Signaling). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).
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