The amount of ELL2 and β-actin was determined by Western blotting analysis. Total protein extracts were prepared with lysis buffer, which was separated on 10% SDS-PAGE and then transferred to the polyvinylidene difluoride membrane (Millipore). The membranes were incubated in 5% nonfat milk powder diluted in PBS containing 0.1% Tween-20 for 2 hours at room temperature and probed with rabbit polyclonal anti-ELL2 antibody (Abcam, ab219865, USA) and anti β-actin monoclonal antibody (Sigma, Japan) in blocking buffer overnight at 4°C. Finally, membranes were incubated with a secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad, USA). Immunocomplexes were detected with the enhanced chemiluminescence method (GE Healthcare, USA).
Enhanced chemiluminescence method
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Enhanced chemiluminescence method is a laboratory technique used to detect and quantify proteins in biological samples. It involves the use of enzymes that emit light when they catalyze a specific chemical reaction, allowing for the visualization and measurement of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Anti gfp, by Merck Group (2 mentions)
Gapdh, by Abcam (2 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Bio-Rad (1 mentions)
Bicinchoninic acid assay kit, by Bio-Rad (1 mentions)
C digit chemiluminescent western blot scanner, by LI COR (1 mentions)
Anti mcherry, by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Bca assay, by Bio-Rad (1 mentions)
Scrib, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Abcam (1 mentions)
β pix, by Merck Group (1 mentions)
Vangl2, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Bca protein assay reagent, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit secondary antibodies, by ZSGB-BIO (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
23 protocols using enhanced chemiluminescence method
1
Western Blotting of ELL2 and β-Actin
2
Protein Expression Analysis in Hippocampal Tissue
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Western blotting was performed to detect the protein expression levels. The hippocampal tissues were homogenized in lysis buffer (10 mM Tris-HCl pH 7.4, 2 mM EDTA, 150 mM NaCl and 0.5% Nonidet P-40) containing protease inhibitors (1 mg/ml leupeptin, 1 mg/ml aprotinin and 1 mg/ml pepstatin A) and incubated for 30 min on ice. Total protein contents of the tissues were determined using the bicinchoninic acid assay kit (Bio-Rad Laboratories, Inc.). Equal amounts of protein (60 µg/lane) from each group were separated by SDS-PAGE (10% gel), and the bands were transferred onto polyvinylidene fluoride membranes (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The membranes were blocked in 3% bovine serum albumin in TBS containing Tween-20 (0.1%; TBST) at room temperature for 40 min and incubated with specific primary antibodies, as described above, at 4°C overnight. The membranes were incubated for 2 h at room temperature with goat anti-mouse horseradish peroxidase-labelled secondary antibody (IgG; cat. no. ab97240; 1:2,000; Abcam, Cambridge, MA, USA) prior to washing with TBST. The enhanced chemiluminescence method (GE Healthcare, Chicago, IL, USA) was used to detect and analyze the immunoreactive bands.
3
Protein Isolation and Western Blot Analysis
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Protein isolation was performed as follows. Isolated heart tissues from rats of all groups were washed with ice-cold PBS followed by minced and homogenization in cold protein lysis buffer and protease inhibitor cocktail (Ansari et al., 2013 ). The cell lysates were incubate on ice for 60 min with vortex mixing after every 10 min, followed by centrifugation for 10 min (12,000 RPM, 4 °C), to obtained total cellular proteins. Total protein content was measured according to the well-established method of Lowry et al. (1951) (link). Western blot analysis was done by following the previously described method of Ansari et al. (2013) . Briefly, protein (25–50 µg) from each group was separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrophoretically moved to nitrocellulose membranes. Protein blots was incubated overnight at 4 °C with primary antibodies against GST, NF-κB p65, pNF-κB p65 and NQO1 and peroxidase-conjugated secondary antibodies at 25 °C. Bands were visualized using the enhanced chemiluminescence method (GE Health Care, Mississauga, Canada). Band intensities were calculated comparative to β-actin bands using an image analysis system (ImageJ® image processing program, National Institutes of Health, Bethesda, USA). Images were capture by using C-Digit chemiluminescent western blot scanner (LI-COR, Lincoln, USA).
4
Western Blot Protein Detection
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Protein samples were resolved in a 12% SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes (GE Healthcare), blocked with 5% skimmed milk in TBST (Tris-buffered saline, 0.1% Tween 20) and probed with anti-GFP (Sigma-Aldrich), anti-mCherry (abcam) or anti-TET8. Then the signals were detected using the enhanced chemiluminescence method (GE Healthcare).
5
Immunoblot Analysis of Apoptosis Signaling
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Cytosolic extracts were prepared from Ramos cells treated with HCS with and without ZDEVD as well as bufalin (10 and 50 nM) for 24 h. Briefly, cells were washed in PBS and then resuspended in 50 μl of lysis buffer [20 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA (ethylenediaminetetraacetic acid), and 1 mM dithiothreitol (DTT)]. After sonication on ice for 3 min with a sonicator 3000 (Misonex Inc., Farmingdale, NY, USA), the protein concentrations were determined by the Bradford assay. Immunoblot assays were performed as per standard procedure. Briefly, equal amounts (50 μg) of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transfer to PVDF membranes. Membranes were probed with the indicated antibodies. Secondary antibodies consisting of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (1:500 vol/vol) were purchased from Santa Cruz. Detection was performed by the enhanced chemiluminescence method from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
6
Western Blot Analysis of BDNF, GABA Signaling
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For western blot studies, the mice (n=3) were anaesthetized with 0.75% isoflurane following 12 hours after last PTZ challenge and were sacrificed by decapitation. The hippocampi were immediately isolated on ice and stored at −80°C until used. To detect the level of expression of BDNF-TrkB, GABAA, and GAD65, western blotting was performed. Hippocampal tissue was homogenized in lysis buffer and incubated on ice for 30 minutes. The protein contents were determined by BCA assay (BioRad, Hercules, CA, USA). The proteins were electrophoreitically separated by SDS-PAGE and were blotted on to PVDF membrane. The membranes were blocked in 3% BSA/TBST at room temperature for 60 minutes and incubated with specific primary antibodies at 4 °C overnight. After washing with TBST, the membranes were further incubated with HRP-labelled secondary antibody (1:2000). The blots were washed further and the immunoreactive bands were detected using an enhanced chemiluminescence method (GE Healthcare, Piscataway, NJ).
7
Western Blot Analysis of Cell Signaling Proteins
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H9C2 cells and mouse hearts were stored at −80°C until use for western blotting.18 (link) Cells were lysed and then the extracts were cleared by centrifugation and stored at −80°C until use. The extracts were boiled in 2× Laemmli sample buffer. Samples were than subjected to SDS–polyacrylamide gel electrophoresis followed by western blot analysis using specific antibodies raised against Scrib (Santa Cruz), Vangl2 (Santa Cruz), Rac1 (Millipore), β-PIX (Millipore), Git1 (Novus Biologicals), or β-tubulin (Abcam). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were used for detection using the enhanced chemiluminescence method (GE Healthcare BioSciences). Quantification of protein levels was determined by densitometry using the ImageJ software. Band intensities were normalized to β-tubulin. The Kruskal–Wallis ANOVA test for non-parametric data was used for statistical analysis.
8
Western Blot Analysis of STAT3 and p-STAT3
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Proteins were extracted from lung tissues using RIPA buffer [Cell Signaling Technology (CST), USA], and the concentrations were quantified using a BCA protein assay reagent (Beyotime, Nanjing, China) according to the manufacturer's instructions. Equal amounts of protein extracts were resolved by 8 or 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were subsequently incubated with primary antibodies, including rabbit anti-mouse STAT3 polyclonal antibodies (1:1,000, CST, USA), rabbit anti-mouse p-STAT3 polyclonal antibodies (1:1,000, CST, USA), and rabbit anti-mouse GAPDH polyclonal antibodies (1:5,000, Proteintech, China) at 4°C overnight. The membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1:5,000, goat, zsgb-bio, China) at room temperature for 1 h. The protein bands were visualized using the enhanced chemiluminescence method (GE Healthcare Life Sciences, Little chalfont, UK) and quantified by using Quantity One software (Bio-Rad, Hercules, CA). GAPDH was used as the internal reference.
9
SDS-PAGE and Western Blotting of C. tentans Cells
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C. tentans epithelial tissue culture cells were boiled in SDS-PAGE sample buffer. Nuclear and cytoplasmic extracts were prepared essentially as previously described (Wurtz et al., 1996 (link)). The protein samples were separated on 12% SDS–polyacrylamide gels and transferred to Immobilon-P polyvinylidene filters (EMD Millipore) by semidry electrophoresis. HRP-labeled secondary antibodies were detected by the enhanced chemiluminescence method (GE Healthcare).
10
Western Blot Analysis of Protein Signaling
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The cells were lysed and the total protein was obtained by using RIPA buffer (Solarbio, Beijing, China). A bicinchoninic acid assay kit (Pierce, Appleton, WI, USA) was used to quantify the total protein. An equal amount of proteins was separated by 12% SDS-PAGE for 40 min and then transferred to PVDF membranes. The membranes were blocked for 1.5 h with 5% nonfat milk to prevent nonspecific binding and then incubated with primary antibodies including anti-SCARA5 (1:1,000; Abcam), anti-E-cadherin (1:1,000; Abcam), anti-N-cadherin (1:1,000; Abcam), anti-p-STAT3 (1:1,000; Abcam), anti-STAT3 (1:1,000; Abcam), anti-p-AKT (1:1,000; Abcam), anti-AKT (1:1,000; Abcam), anti-PI3K (1:1,000; Abcam), and p-PI3K (1:1,000; Abcam) at 4℃ overnight. In the next day, the membranes were incubated with secondary antibody for 2 h. The protein bands were visualized by enhanced chemiluminescence method (GE Healthcare Life Sciences, Piscataway, NJ, USA). GAPDH (1:1,000, Abcam) was served as the loading control for normalization.
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