Brain tissues were collected and divided into the cerebrum and cerebellum. Tissues were homogenized in a grinder in radioimmunoprecipitation assay (RIPA) buffer on ice for 30 minutes. After centrifugation at 10,000 ×g for 15 minutes of tissue lysate, the protein concentration of supernatant was determined by protein assay dye (Bio-Rad, Hercules, CA, USA). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (PerkinElmer, Waltham, MA, USA). After blocking, the membranes were incubated with primary antibodies, which including connexin 36 (Cx36) and phosphorylated Ca2+/calmodulin-dependent protein kinase (p-CaMKII, Santa Cruz Biotechnology, Santa Cruz, CA, USA), synaptophysin (SYP, Proteintech, Rosemont, IL, USA), and β-actin (Sigma), at 4°C for 16 h. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). The membranes were developed using enhanced chemiluminescence reagents (Perkin Elmer, Waltham, MA, USA).
Enhanced chemiluminescence reagent
Manufactured by PerkinElmer
Sourced in United States, United Kingdom
Enhanced chemiluminescence reagents are laboratory products that generate a luminescent signal upon the occurrence of a chemical reaction. These reagents are commonly used in various biochemical and molecular biology applications to detect and quantify the presence of specific proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (15 mentions)
Quantity one 4, by Bio-Rad (11 mentions)
Bca protein assay, by Thermo Fisher Scientific (9 mentions)
Ripa buffer, by Merck Group (8 mentions)
Coomassie protein assay reagent, by Thermo Fisher Scientific (6 mentions)
β actin, by Merck Group (5 mentions)
Quantity one analysis software, by Bio-Rad (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Las 4000, by Cytiva (3 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Rabbit anti rat ace2, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti rat at2r, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Molecular imager, by Bio-Rad (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
107 protocols using enhanced chemiluminescence reagent
1
Western Blot Analysis of Brain Proteins
2
Quantifying Nociceptor Protein Expression
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Bilateral L4 and L5 DRGs were quickly removed from deeply anesthetized rats and stored at 80°C. Sequential precipitation procedures were used on the tissue samples that were lysed in ice-cold (4°C) NP-40 lysis buffer containing a mixture of protease inhibitor, phosphatase inhibitors, and phenylmethylsulfonyl fluoride (Sigma-Aldrich). The protein concentrations of the lysates were quantified using the BCA method (with reagents from Pierce), and the total protein content between samples was equalized. Protein samples were separated by SDS-PAGE and transferred to PVDF membrane (both from Bio-Rad Laboratories). The following primary antibodies were used: anti-P2X3 (1 : 1000; Neuromics, Edina, MN, USA), anti-TRPV1 (1 : 100; Santa Cruz), and β-actin (1 : 2000; Bioworld, St. Louis Park, MN, USA). The membranes were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) with horseradish peroxidase-conjugated secondary antibodies (R&D Systems, Minneapolis, MN, USA). Data were analyzed with ImageJ.
3
Western Blot Analysis of Angiotensin Receptors
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Western blot analysis was performed as we described previously (Tain et al., 2014 (link)). We used the following antibodies: for angiotensin converting enzyme 2 (ACE2), rabbit anti-rat ACE2 (1:1000, overnight incubation; Santa Cruz Biotechnology, Santa Cruz, California, USA); for angiotensin II type 2 receptor (AT2R), rabbit anti-rat AT2R (1:250, overnight incubation; Santa Cruz Biotechnology); for angiotensin (1–7) receptor MAS, a rabbit anti-rat MAS antibody (1:1000, overnight incubation; Santa Cruz Biotechnology). Bands of interest were visualized using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA) and quantified by densitometry (Quantity One Analysis software; Bio-Rad), as integrated optical density (IOD) after subtraction of background. The IOD was factored for Ponceau red staining to correct for any variations in total protein loading. The protein abundance was represented as IOD/PonS.
4
Western Blot Analysis of ER Stress and Signaling Pathways
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Samples (cells or spinal cord tissue segments at L1-L6) were collected and washed with ice-cold PBS before being lysed in radio immunoprecipitation assay (RIPA) lysis buffer and then sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 10 % low-fat dry powdered milk or with 5% BSA and 5% low-fat dry powdered milk in TBST (Tris–HCl, NaCl, Tween 20) for 2 h at room temperature, and then probed with primary antibodies at 4°C overnight. Finally, the horseradish peroxidase (HRP)-coupled secondary antibodies were utilized for detecting corresponding primary antibody. The primary antibodies utilized included β-actin (1:5000), Caspase-12 (1:1000), HSP70 (1:1000), GRP78 (1:500), ATF6 (1:1000), p-eIF2α (Ser51) (1:1000), eIF2α (1:1000), IRE1α (1:1000), XBP1s (1:1000), ERK (1:1000), p-ERK (Thr202/Tyr204) (1:1000), PKA (1:1000), p-PKA(Thr197) (1:1000), NMDA Receptor subunit 1 (NR1) (1:1000), and Phospho-NMDA Receptor subunit 1 (p-NR1) (Ser897) (1:1000). The bands were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, United States). Data were analyzed with the Molecular Imager and the associated software Image J (NIH, United States). The original unprocessed images of all western blots were provided in Supplementary Data Sheets S1 –S10 .
5
Evaluating Protein Expression in Cancer Cell Lines
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According to the changes in gene expression, some proteins associated with cell invasion were detected by Western blot. A2780 cells were treated with 32 and 64 μM DHA; HO8910 cells were treated with 60 and 120 μM DHA; and SKOV-3 cells were treated with 30 and 60 μM DHA. After 72 hours, cells were washed with ice-cold PBS and cracked in RIPA lysis buffer (Beyotime, Haimen, China) containing protease inhibitor (Complete, Roche Diagnostics, Mannheim, Germany). Cell lysate was centrifuged at 13,000 rpm for 10 minutes at 4°C; the supernatant was collected and boiled at 100°C for 10 minutes. Protein concentrations were measured using the BCA protein assay kit. Then, 50 μg of proteins was electrophoresed in SDS-PAGE, and transferred onto PVDF (polyvinylidene difluoride) membranes in a wet-transfer apparatus. Membranes were blocked with 5% bovine serum albumin in TBS-Tween for 1 hour and then incubated with a specific primary antibody (VEGF, COX-2, MMP-3, MMP-9, N-Ca, WAVE3, STAT3, and NF-κB) overnight at 4°C. Membranes were washed 3 times with TBS-Tween (TBS containing 0.1% Tween 20) and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature, and blots were developed with enhanced chemiluminescence reagents (PerkinElmer Life Sciences, Waltham, MA) and exposed to x-ray film. The expression levels were normalized to GAPDH.
6
Protein Expression Analysis in Ankle and Spinal Cord
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Periarticular tissue of the ankle and the spinal cord segments at L1-L6 were rapidly removed and homogenized in RIPA Lysis Buffer after the animals’ deep anesthesia with chloral hydrate. The protein concentrations were determined by BCA Protein Assay (Thermo Fisher, Waltham, MA), and 30–60 μg of proteins were loaded and separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA). The membranes were blocked with 5% bovine serum albumin for 2 h at room temperature, probed with antibodies overnight at 4 °C with the primary antibodies, and then incubated with HRP-coupled secondary antibodies. The primary antibodies used included IL-1β (1:500), p-NR1 (1:1000), p-p38 (1:1000), p-JNK (1:1000), p-ERK (1:1000), GAPDH (1:8000), NLRP3 (1:1000), and caspase-1 (1:1000). The filters were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA) with secondary antibodies (Chemicon, Billerica, MA). Data were acquired with the Molecular Imager (Gel DocTM XR, 170-8170) and analyzed with Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA, USA).
7
Western Blot Analysis of Protein Signaling
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Tissues were lysed in RIPA-buffer (Sigma–Aldrich, MO, USA) using sonicator (Diagnode, Seraing, Belgium) and centrifuged (12,000 g, 10 min, 4 °C) [21] . Proteins in supernatants were collected and blotted for anti-phospho AKTSer473 (Cat# 9271), anti-AKT (Cat# 9272), anti-phospho AMPKThr172 (Cat# 2531), anti-AMPK (Cat# 2532), anti-Actin (Cat# 12,262), anti-GAPDH (all Cell Signaling Technologies, Cell Signaling Technology, Danvers, MA, USA), anti-UCP1 (Abcam, Cambridge, UK, ab10983) and anti-GLUT1 (Dr. A. Schürmann, Potsdam) diluted in 1x TTBS+5% BSA and incubated overnight at 4 °C under gentle agitation. Subsequently, blots were incubated with HRP-conjugated goat anti-rabbit IgG secondary antibody (Dianova, Hamburg, Germany) diluted 1:20,000 in 1x TTBS+5% BSA for 1h at room temperature. Specific protein bands were visualized using enhanced chemiluminescence reagents (Perkin Elmer, Waltham, MA, USA). Protein bands were quantified using Quantity One software (BioRad, München, Germany).
8
Western Blot Protein Analysis Protocol
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Tissues were lysed using mortar and pestle, resuspended in RIPA buffer (1% Triton, 1% deoxycholate, 0.1% SDS, 0.16M NaCl, 10 mmol/L Tris pH 7.4, and 5 mmol/L EDTA), supplemented with a protease inhibitor cocktail (Pharmingen, San Diego, CA, USA), and analyzed as described previously (48). Primary antibodies used for DEK were as follows: DEK (1:1000; BD Biosciences, San Diego, CA, USA), pan-actin (1:20,000; a gift from James Lessard). Membranes were exposed to enhanced chemiluminescence reagents (Perkin Elmer, Boston, MA, USA) and imaged using the BioRad Chemidoc (Hercules, CA, USA).
9
Western Blot Analysis of Spinal Cord Proteins
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The spinal cord segments at L1-L6 were rapidly removed and homogenized in RIPA Lysis Buffer after the animals deep anesthesia with chloral hydrate [22 (link)]. The protein concentrations were determined by BCA Protein Assay (Thermo Fisher, Waltham, MA), and 40 μg of proteins (unless specified otherwise) was loaded and separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore Corp., Bedford, MA). The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature, probed with antibodies overnight at 4 °C with the primary antibodies, and then incubated with HRP-coupled secondary antibodies. The primary antibodies used included IL-1β (1:500), p-NR1(1:1000), p-PKCγ (1:1000), p-p38 (1:1000), and IBA-1 (1:1000). For loading control, the blots were probed with antibody for GAPDH (1:8000). The filters were then developed by enhanced chemiluminescence reagents (PerkinElmer) with secondary antibodies (anti-rabbit or anti-mouse or anti-goat antibody, 1:1000) (Chemicon). Data were analyzed with the Molecular Imager (Gel DocTM XR, 170-8170) and the associated software Quantity One-4.6.5 (Bio-Rad Laboratories, Berkeley, CA).
10
Western Blot Analysis of Spinal Cord Tissue
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Samples (cells or spinal cord tissue segments at L1-L6) were collected and washed with ice-cold PBS before being lysed in radio immunoprecipitation assay (RIPA) lysis buffer and then sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene uoride membranes (Millipore). The membranes were blocked with 10 % low-fat dry powdered milk or with 5% BSA and 5% low-fat dry powdered milk in TBST (Tris-HCl, NaCl, Tween 20) for 2 h at room temperature, and then probed with primary antibodies at 4°C for overnight. Finally, the horseradish peroxidase (HRP)-coupled secondary antibodies were utilized for detecting corresponding primary antibody. The primary antibodies utilized included β-actin (1:5000), AMPK (1:1000), p-AMPK (1:1000) (Thr172), HO-1 (1:1000), NF-κB p65 (1:1000), p-NF-κB p65 (Ser536) (1:1000), HMGB1 (1:1000), Transferrin (1:1000) and IL-1β (1:300). The bands were then developed by enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA). Data were analyzed with the Molecular Imager and the associated software Image J (NIH, USA).
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