Total protein was isolated from approximately 50 mg of snap-frozen 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 (1:250; Millipore, Billerica, MA), CRF2 (1:1000; Millipore), 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 (1:2000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).
Criterion blotter wet transfer
Manufactured by Bio-Rad
Sourced in Germany
The Criterion Blotter is a wet transfer system designed for protein transfer from polyacrylamide gels to membrane supports. It facilitates the efficient and consistent transfer of proteins from SDS-PAGE gels to PVDF or nitrocellulose membranes.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit secondary antibody, by Cell Signaling Technology (6 mentions)
Quantity one 4, by Bio-Rad (6 mentions)
Halt protease and phosphatase inhibitor, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Cell Signaling Technology (6 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (5 mentions)
Dc protein assay, by Thermo Fisher Scientific (5 mentions)
Na3vo4, by Merck Group (5 mentions)
Criterion 4 to 12 bis tris gels, by Bio-Rad (4 mentions)
Trpv1, by Alomone (3 mentions)
Trpa1, by Aviva Systems Biology (3 mentions)
Criterion 4 12 bis tris gels, by Bio-Rad (2 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
6 protocols using criterion blotter wet transfer
1
Colon Protein Extraction and Western Blot
2
Protein Extraction and Western Blot Analysis
3
Protein Extraction and Western Blot Analysis
4
Protein Expression Analysis in DRG, Vagina, and Colon
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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).
5
Colon Protein Extraction and Immunoblotting
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Total protein was isolated from approximately 50 mg of snap-frozen 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 min in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4%–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 h at room temperature in 5% milk in Tris-buffered saline with Tween-20 (TBST) and incubated overnight at 4 °C with antisera to CRF1 (1:250; Millipore, Billerica, MA), CRF2 (1:1000; Millipore), 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 h with anti-rabbit secondary antibody (1:2000; Cell Signaling, Danvers, MA). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad).
6
Dural Protein Extraction and Western Blot Analysis
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Mice were overdosed with inhaled isoflurane (<5%) and dura was removed and flash frozen in liquid nitrogen before storage at −80°C. Dural protein was isolated using Cell Extraction Buffer containing Halt protease and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA, United States) and Na3VO4. Protein concentrations were determined using a Pierce BCA assay (Thermo-Fisher Scientific, Waltham, MA, United States). Samples were reduced by heating to 95°C in the presence of 2-mercaptoethanol, subjected to SDS-PAGE (Criterion 4–12% Bis-Tris gels; Bio-rad laboratories), and transferred to Nitrocellulose transfer membrane (Whatman GmbH, Dassel, Germany) by Criterion Blotter wet transfer (Bio-Rad). The membranes were blocked for 1 h in 5% milk in Tris-buffered saline with Tween-20 (TBST) then incubated overnight with protease-activated receptor 2 (PAR2; 1:1000; Abcam), transient receptor potential ankyrin 1 (TRPA1;1:1000; Aviva) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH;1:2000; Cell Signaling) antisera. Membranes were washed with TBST and incubated for 1 h with anti-rabbit secondary antibody (1:10,000; Cell Signaling). Densitometry was performed using Quantity One 4.6.9 software (Bio-Rad Laboratories). PAR2 and TRPA1 protein levels were normalized to GAPDH.
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