Cells were washed with sterile phosphate buffered saline and lysed with Radioimmunoprecipitation assay buffer buffer. The lysate containing equal amount of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to an Immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were then incubated with specific primary antibodies and then incubated with corresponding secondary antibody conjugated with horseradish peroxidase. Immunoblot signals were developed by enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL, USA), and analyzed using an ImageQuant™ LAS 4000 biomolecular imager (GE Healthcare Life Sciences, Waukesha, WI, USA) with Multi Gauge 3.0 software (Fujifilm Life Science, Tokyo, Japan). Antibodies used are as follows: Phospho-p38(p-p38), p-JNK, p-ERK, p38, JNK, ERK and p65 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-TNFα and anti-HMGB1 antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-β-actin and lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Immune blot polyvinylidene difluoride pvdf membrane
Manufactured by Bio-Rad
Sourced in United States
The Immune-Blot™ polyvinylidene difluoride (PVDF) membrane is a laboratory product designed for protein transfer and detection applications. It is a chemically and mechanically stable membrane material that can be used for Western blotting and other protein analysis techniques.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated anti mouse and anti rabbit igg, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Multi gauge 3, by Fujifilm (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Anti hmgb1 antibody, by Abcam (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 biomolecular imager, by GE Healthcare (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti tnf α, by Abcam (1 mentions)
Las 4000, by Cytiva (1 mentions)
Supersignal west dura extended duration chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Cl xposure film, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay ripa buffer, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Criterion tgx 4 20 gels, by Bio-Rad (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
5 protocols using immune blot polyvinylidene difluoride pvdf membrane
1
Western Blot Analysis of Signaling Proteins
2
Western Blotting Protocol for Protein Analysis
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Protein lysates from homogenised tissues and cultured cells were extracted using a Radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, Dorset, UK) supplemented with protease inhibitor (Sigma-Aldrich, Dorset, UK) and phosphatase inhibitor (PhosSTOP, Roche Diagnostics Ltd., Burgess Hill, UK). Lysates were analysed by SDS-PAGE under reducing conditions on precast 12% gels (Mini-PROTEAN TGX, Bio-Rad, Hertfordshire, UK) and transferred to an Immune-Blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hertfordshire, UK). The primary antibody sources, concentrations, and incubation conditions are detailed in Table S3 . Horseradish peroxidase (HRP)-linked secondary antibodies were from Thermo Fisher Scientific (Loughborough, UK). Signal detection was performed using SuperSignal West Dura Extended Duration chemiluminescent Substrate (Thermo Fisher Scientific, Loughborough, UK) and CL-Xposure film (Thermo Fisher Scientific, Loughborough, UK).
3
Western Blot Analysis of Hepatitis B Virus X Antigen
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Bacteria were lysed using a lysis Buffer solution (1% Triton X-100, 50 mM Tris-HCl, 150 mM NaCl, 0.25% sodium deoxycholate, 1 mM Ethylene glycol tetraacetic acid, and 1 mM NaF) containing 1 mM Na3VO4, 1 mM phenylmethanesulfonyl fluoride or phenylmethylsulfonyl fluoride and protease inhibitor pellet [16, 17, 19] .
To western blotting, the lysates were mixed with the same volume of 5X Laemmli sample buffer. Bradford assay was used to measure protein quantitation. After a 5 min boiling, 15 μg lysates were subjected to SDS-PAGE. They were then transferred to a 0.2 μm immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA). The 5% BSA (Cat No: A-7888; Sigma Aldrich, MO, USA) was used to block the membranes for 1 h (in 0.1% Tween 20). The membranes were then incubated together with anti-Hepatitis B virus X antigen-antibody (Cat. No: ab39716-Abcam antibody) for 1 h at room temperature. The membranes were then rinsed with TBST, and incubated with goat anti-rabbit IgG H&L (HRP) (Cat No: ab205718-Abcam antibody) as a secondary antibody. Finally, the membranes were placed in an incubator with enhanced chemiluminescence (ECL) for 1-2 minutes [16] [17] [18] [19] [20] .
To western blotting, the lysates were mixed with the same volume of 5X Laemmli sample buffer. Bradford assay was used to measure protein quantitation. After a 5 min boiling, 15 μg lysates were subjected to SDS-PAGE. They were then transferred to a 0.2 μm immune-Blot™ polyvinylidene difluoride (PVDF) membrane (Cat No: 162-017777; Bio-Rad Laboratories, CA, USA). The 5% BSA (Cat No: A-7888; Sigma Aldrich, MO, USA) was used to block the membranes for 1 h (in 0.1% Tween 20). The membranes were then incubated together with anti-Hepatitis B virus X antigen-antibody (Cat. No: ab39716-Abcam antibody) for 1 h at room temperature. The membranes were then rinsed with TBST, and incubated with goat anti-rabbit IgG H&L (HRP) (Cat No: ab205718-Abcam antibody) as a secondary antibody. Finally, the membranes were placed in an incubator with enhanced chemiluminescence (ECL) for 1-2 minutes [16] [17] [18] [19] [20] .
4
Protein Expression Analysis in Intestinal Cells
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Primary enteric neuronal cells and intestinal Caco-2 cells, along with supernatant, were lysed in 1× Laemmli samples loading buffer (Bio-Rad, Hercules, CA, USA), supplemented with a complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), and proteins separated on Criterion TGX 4-20% gels (Bio-Rad) according to recommended procedure. Separated proteins were transferred onto Immune-Blot polyvinylidene difluoride (PVDF) membranes (Bio-Rad), according to recommended procedure. The membranes were then probed with anti-BiP (rabbit, #3177, 1:1000, Cell Signaling Technology, Boston, MA, USA), anti-HSP70 (rabbit, #4872, 1:1000, Cell Signaling Technology, Boston, MA, USA), anti-CD91 (rabbit, #26387, 1:1000, Cell Signaling Technology, Boston, MA, USA), and anti-β-actin (mouse, #a5441, 1:5000, Sigma-Aldrich, St. Louis, MO, USA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology) secondary antibodies were used at a 1:2000 dilution. All semi-quantitative measurement of band intensity was performed using the ImageJ analysis software (US National Institutes of Health, Bethesda, MD, USA).
5
Western Blot Analysis of Neuronal Proteins
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Neuronal cells were lysed in 1 × Laemmli sample loading buffer (Bio-Rad, Hercules, CA, USA) supplemented with complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and the proteins separated on 4–20% Criterion TGX gradient gels (Bio-Rad) according to recommended procedure. Separated proteins were transferred onto Immune-Blot polyvinylidene difluoride (PVDF) membranes (Bio-Rad) according to recommended procedure. The membranes were then probed with primary rabbit antibodies to nNOS (#ab78078, Abcam), cleaved caspase-1 (#4199, Cell Signaling Technology, Boston, MA), SIRT3 (#5490, Cell Signaling Technology) diluted 1:1000, and mouse monoclonal antibodies to α-tubulin (#3873S, Cell Signaling Technology) and β-actin (#a5441, Sigma-Aldrich, St. Louis, USA) diluted, respectively, 1:1000 and 1:5000. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG (Cell Signaling Technology) secondary antibodies were used at a 1:2000 dilution. All semi-quantitative measurement of band intensity was performed using the ImageJ analysis software (US National Institutes of Health, Bethesda, Maryland, USA).
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