Nuclear factor of activated T cell 5 (NFAT5) expression was assessed using the western blot assay, as previously described (21 (link)). Briefly, CTX-TNA2 cells were harvested and lysed in RIPA buffer (Beyotime, Shanghai, China). The mixture supernatants were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels and were then transferred onto polyvinylidene fluoride membranes. The membranes were incubated overnight with NFAT5 monoclonal antibody (dilution, 1:1000, ab137407, Abcam, San Diego, CA, USA) and GAPDH loading control monoclonal antibody (dilution, 1:10,000, MA5-15738, Ebioscience, San Diego, CA, USA). The bound antibodies were detected using horseradish peroxidase-conjugated goat anti-mouse/rabbit IgG secondary antibody (dilution, 1:10,000, G-21040/21234, Ebioscience) and visualized using the enhanced chemiluminescent reagent (PerkinElmer Life Sciences, MA, USA).
Enhanced chemiluminescent reagent
Manufactured by PerkinElmer
Sourced in United States
Enhanced chemiluminescent reagent is a laboratory product used to detect and quantify proteins in Western blotting applications. It is designed to generate a luminescent signal in the presence of the target protein, allowing for sensitive and accurate detection.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (2 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab137407, by Abcam (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Ab128915, by Abcam (1 mentions)
Ab68183, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Mes running buffer, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Merck Group (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Ab47405, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
X ray film, by Carestream (1 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Antibody against phospho ampk thr172, by Cell Signaling Technology (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
X ray film, by Merck Group (1 mentions)
2 β mercaptoethanol, by Merck Group (1 mentions)
11 protocols using enhanced chemiluminescent reagent
1
Quantifying NFAT5 Expression by Western Blot
2
Protein Expression Analysis of Cerebellum in BTBR and WT Mice
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The cerebellum of BTBR and WT mice were dissected and snap-frozen in dry ice. Brain tissues were homogenized in ice-cold lysis buffer (20 mM HEPES pH7.5, 100 mM NaCl, 0.05% Triton X-100, 1 mM DTT, 5 mM Na-Betaglycerophosphate, 0.5 mM Na-vanadate, 1 mM EDTA, protease inhibitors) and protein was extracted and quantified by Bradford assay. Fifty microgram protein lysates were resolved on a 10% SDS polyacrylamide gel and transferred to PVDF membrane. Membranes were blocked by 5% non-fat dry milk and incubated with primary antibodies overnight at 4 °C. Next day blots were washed and incubated in horseradish peroxidase (HRP) conjugated with anti-rabbit or anti-mouse IgG antibodies (1:5000; Bio-Rad) for 1.5 h and washed again. To detect immunoreactivity, blots were incubated with enhanced chemiluminescent reagent (Perkin Elmer) and imaged on Odyssey Fc Imaging System (LI-COR Biosciences). Relative intensity of blots was quantified using ImageJ-NIH software.
Primary antibodies were: anti-Kv1.2 (1:1000; SAB5200059), anti-FMRP (1:100; NBP2-01770), anti-pFMRP (1:50; ab48127), anti-mTOR (1:1000; 2983S), anti-p-mTOR (1:1000; 2971S), anti-ERK (1:1000; sc-514302), anti-p-ERK (1:1000; 9101S), anti-mGluR5 (1:100; PA1-38132), and anti-β-actin (1:5000; A5441).
Primary antibodies were: anti-Kv1.2 (1:1000; SAB5200059), anti-FMRP (1:100; NBP2-01770), anti-pFMRP (1:50; ab48127), anti-mTOR (1:1000; 2983S), anti-p-mTOR (1:1000; 2971S), anti-ERK (1:1000; sc-514302), anti-p-ERK (1:1000; 9101S), anti-mGluR5 (1:100; PA1-38132), and anti-β-actin (1:5000; A5441).
3
Western Blot Analysis of β-Catenin
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The extractive total proteins were separated by performing SDS-PAGE using 10% resolving gels, then transferred onto polyvinylidene fluoride membranes and incubated with rabbit monoclonal antibody specific to β-catenin (dilution, 1:500, ab68183, Abcam, San Diego, CA, USA) and rabbit monoclonal antibody specific to GAPDH (dilution, 1:10,000, ab128915, Abcam) for 40 min. The membranes were washed three times and incubated with Goat Anti-Rabbit IgG H&L (HRP) (dilution, 1:10,000, ab205718, Abcam). Finally, protein bands were visualized using an enhanced chemiluminescent reagent (PerkinElmer Life Sciences, MA, USA).
4
Western Blot Analysis of Aspirin Effect
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Cells were seeded and treated as above, except fresh aspirin-containing medium was added 1 h before harvesting, as previously described [20 (link)]. Whole cell lysates were harvested on days 1 and 4 with and without aspirin in Laemmli Buffer (Bio Rad, Hercules, CA) and boiled for 10 min at 100 °C. Lysates were resolved using SDS-PAGE using 4–12 % bis–tris NuPAGE gels in MES running buffer (Invitrogen, Grand Island, NY) following the manufacturer’s protocol. The proteins were transferred using Invitrogen Xcell II blotting apparatus to a PVDF membrane (Invitrogen, Grand Island, NY). Following transfer, the membranes were blocked in 5 % w/v milk in tris(hydroxymethyl)aminomethane (TRIS)-buffered saline supplemented with 0.1 % tween-20 (Sigma, Saint Louis, MO) for 1 h. Membranes were probed with either phosphorylated GSK3β (Ser 9; 9336), total GSK3β (9315), phosphorylated Src family (Tyr 416; 2101), total Src (ab47405, Abcam, Cambridge, MA), ACTB (β-actin) (4967), and TUBB (β-tubulin) (2146) primary antibody followed by incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (7074). Protein bands were visualized using enhanced chemiluminescent reagent (Perkin-Elmer, Waltham, MA). All antibodies were purchased from Cell Signaling Technology (Beverly, MA) unless otherwise noted. Densitometry was performed using Image J analysis software (NIH).
5
Western Blot Protein Extraction and Analysis
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To prepare tissue lysates, freshly dissected tissues from mice were placed in ice-cold RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, 1 mM EDTA, pH 7.5) and homogenized using a glass homogenizer and centrifuged at 13,200 rpm for 5 min. Supernatants were collected and mixed with an equal volume of 2 3 SDS-PAGE loading buffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, 2% mercaptoethanol, 0.1% bromophenol blue, pH 6.8) and boiled for 10 min. Cultured cells were directly lysed in 1 3 SDS-PAGE loading buffer and boiled.
Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (General Electric Company). Blots were preblocked with 5% non-fat milk diluted in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 hr and then incubated with primary antibodies. After thorough washing with TBST, membranes were incubated with secondary IgG-HRP antibodies. After extensive wash in TBST, protein bands were visualized with enhanced chemiluminescent reagent (PerkinElmer) and exposed to X-ray films (Carestream) or Mini Chemiluminescent Imager (MiniChemi 610 Plus, Beijing Sage Creation Science Co).
Proteins separated by SDS-PAGE were transferred to nitrocellulose membranes (General Electric Company). Blots were preblocked with 5% non-fat milk diluted in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.5) for 1 hr and then incubated with primary antibodies. After thorough washing with TBST, membranes were incubated with secondary IgG-HRP antibodies. After extensive wash in TBST, protein bands were visualized with enhanced chemiluminescent reagent (PerkinElmer) and exposed to X-ray films (Carestream) or Mini Chemiluminescent Imager (MiniChemi 610 Plus, Beijing Sage Creation Science Co).
6
Quantifying USP48 and GAPDH Levels by Western Blotting
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USP48 and GAPDH protein levels were analyzed by western blotting [24 (link)]. Briefly, KNG cells were lysed, and 30 µg of protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Subsequently, all membranes were blocked with 5% skimmed milk diluted in TBS at 37˚C for 1 h and washed thrice with TBST. The membranes were then incubated with rabbit polyclonal USP48 (dilution 1:500, ab237765, Abcam, San Diego, CA, USA) and rabbit monoclonal antibodies against GAPDH (dilution 1:10,000, AF7021, Affinity) at 4˚C for 12 h. The membranes were washed thrice with TBST. Secondary antibodies were detected using Goat Anti-Rabbit IgG H&L (HRP) (dilution, 1:10,000, ab205718, Abcam) and visualized using an enhanced chemiluminescent reagent (PerkinElmer Life Sciences, MA, USA).
7
Western Blot Analysis of Phospho-AMPK
8
Western Blot Protein Expression Analysis
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Protein expression was determined by a western blot assay. Protein samples were separated and resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with primary antibody at 4°C overnight, followed by a second incubation with a horseradish peroxidase (HRP)-conjugated secondary immunoglobulin G (IgG) antibody; immunoreactive bands were visualized with PerkinElmer (Waltham, MA, United States) enhanced chemiluminescent reagents (Yu et al., 2011 (link)).
9
Western Blot Protein Analysis Protocol
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Protein samples were separated and resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with a primary antibody at 4 °C overnight, and then incubated with a horseradish peroxidase (HRP)-conjugated secondary immunoglobulin G (IgG) antibody; immunoreactive bands were visualized with PerkinElmer enhanced chemiluminescent reagents [44 (link)].
10
Quantitative Western Blot Analysis
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The procedures of Western blot were carried out as previously described [61 (link)]. Protein samples were prepared in RIPA buffer and protein concentrations were determined using the Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). Equal amounts of protein samples (25 µg) were separated in a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (#66485; PALL Life Sciences) via semi-dry transfer (350 mA, 90 min). Blots were blocked with 5% skim milk in tris-buffered saline with 0.1% tween-20 (TBST), and incubated with primary at 4 ºC overnight and with corresponding horseradish peroxidise (HRP) and then corresponding secondary antibodies at room temperature. Protein signals of interest were visualized using enhanced chemiluminescent reagents (PerkinElmer, Mass, USA) and analyzed by exposure to film (HyBlot CL, NJ, USA). Densitometry was employed to quantify proteins of interest using the gel analyzer function of ImageJ (ver. 1.46a; National Institutes of Health, USA).
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