The protein levels were validated using a Rat-Brain-Derived Neurotrophic Factor ELISA Kit (E0476Ra; Bioassay Technology Laboratory, Shanghai, China) and a Rat Tyrosine Kinase B ELISA Kit (E1598Ra; Bioassay Technology Laboratory, Shanghai, China) following the manufacturers’ protocols. Briefly, HIP was homogenized in cold PBS (pH 7.4) with cocktails of protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MI, USA) and centrifuged for 5 min at 5000× g. A bicinchoninic acid assay (BCA) protein assay kit (Serva, Heidelberg, Germany) was used for protein concentration measurement in the supernates. The absorbance of duplicates of each sample and the standards were measured at a wavelength of λ = 450 nm using a Multiskan Spectrum spectrophotometer (Thermo LabSystems, Philadelphia, PA, USA). The concentration of proteins was calculated from standard curves and expressed as ng/mg of protein.
Cocktails of protease and phosphatase inhibitors
Manufactured by Merck Group
Sourced in United States
Cocktails of protease and phosphatase inhibitors are a type of lab equipment used to protect proteins from degradation during sample preparation and analysis. These inhibitor cocktails contain a combination of chemical compounds that effectively block the activity of proteases and phosphatases, enzymes that can break down or modify proteins. The core function of these inhibitor cocktails is to preserve the integrity and native state of proteins, enabling researchers to accurately study and analyze their structure, function, and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (3 mentions)
Dc protein assay kit, by Bio-Rad (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Image lab 3, by Bio-Rad (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Multiskan spectrum spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Secondary goat anti mouse and anti rabbit antibodies, by Santa Cruz Biotechnology (1 mentions)
Gradient gel, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Bca protein assay kit, by Serva Electrophoresis (1 mentions)
Bioprep 24, by Allsheng (1 mentions)
Ab87811, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Genechip rat gene 2.0 st array, by Thermo Fisher Scientific (1 mentions)
Reagents for sds page, by Merck Group (1 mentions)
Phospho creb ser133 87g3 rabbit mab, by Cell Signaling Technology (1 mentions)
Bax rabbit pab, by Cell Signaling Technology (1 mentions)
Genechiph wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
β actin d6a8 rabbit mab, by Cell Signaling Technology (1 mentions)
10 protocols using cocktails of protease and phosphatase inhibitors
1
Quantifying Brain-Derived Neurotrophic Factors
2
Protein Extraction and Immunoblotting
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Cells were harvested in lysis buffer consisting
of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100, 1 mM
EDTA, 1 mM EGTA, and cocktails of protease and phosphatase inhibitors
(Sigma-Aldrich, St. Louis, MO). Cell lysates were clarified by centrifugation
for 5 min, and the protein concentration of the supernatants was determined
using a modified Bradford assay (Bio-Rad, Hercules, CA). For immunoblotting,
20 μg of protein was loaded in each lane and was separated by
SDS–PAGE on 4–12% gradient gels (Invitrogen, Carlsbad,
CA), transferred to PVDF membranes and detected by immunoblotting
with the following primary antibodies. Goat anti-mouse and anti-rabbit
secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated
to horseradish peroxidase were used at a 1:3000 dilution, and immunoreactive
bands were detected by chemiluminescence (SuperSignal, Pierce, Rockford,
IL) and film (Denville Scientific, South Plainfield, NJ).
of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 1% Triton X-100, 1 mM
EDTA, 1 mM EGTA, and cocktails of protease and phosphatase inhibitors
(Sigma-Aldrich, St. Louis, MO). Cell lysates were clarified by centrifugation
for 5 min, and the protein concentration of the supernatants was determined
using a modified Bradford assay (Bio-Rad, Hercules, CA). For immunoblotting,
20 μg of protein was loaded in each lane and was separated by
SDS–PAGE on 4–12% gradient gels (Invitrogen, Carlsbad,
CA), transferred to PVDF membranes and detected by immunoblotting
with the following primary antibodies. Goat anti-mouse and anti-rabbit
secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated
to horseradish peroxidase were used at a 1:3000 dilution, and immunoreactive
bands were detected by chemiluminescence (SuperSignal, Pierce, Rockford,
IL) and film (Denville Scientific, South Plainfield, NJ).
3
Quantitative Analysis of MMP-9, D1R, and Cav1.2 in Rat Brain
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Quantitative measurement of MMP-9, D1R, and Cav1.2 in such rat brain structures as the vSTR was performed using a Rat MMP-9 ELISA Kit (Reddot Biotech, Kelowna, BC, Canada), a Rat Dopamine Receptor D1 (DRD1) ELISA kit (Reddot Biotech, Kelowna, BC, Canada), and Rat Voltage-dependent L-type Calcium Channel Subunit Alpha-1C ELISA Kit (Bioassay Technology Laboratory, Shanghai, China), respectively, following manufacturers’ protocols. Firstly, frozen brain structures were homogenized in cold buffer at pH 7.4 (0.32 M sucrose, 1 mM HEPES, 1 mM MgCl2, 1 mM NaHCO3, and 0.1 mM PMSF) containing cocktails of protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, MO, USA), using a homogenizer ball (Bioprep-24, Allsheng, China) (10 s at 10,000 rpm). Then, homogenates were centrifuged for 5 min at 5000× g, the supernates were immediately removed, and protein concentration in the supernates was measured using a bicinchoninic acid assay (BCA) protein assay kit (Serva, Heidelberg, Germany). From each sample, 100 µg of protein was used in each ELISA assay. All data are expressed in ng/mL.
4
Protein Expression Analysis by Western Blot
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Whole protein extracts were lysed in ice-cold RIPA lysis buffer containing cocktails of protease and phosphatase inhibitors (Sigma) according to the manufacturer's protocol. Total proteins from each lysate were separated by SDS-PAGE and transferred onto PVDF membranes and then blocked with 5% nonfat milk for 1 hour. The membranes were then probed with the indicated primary antibodies at 4°C with gentle shaking overnight and incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies. Then, the proteins were visualized by chemiluminescence, and signals were quantified by Image J software. Antibodies used in this study are listed in Table 1 .
5
Western Blot Analysis of Signaling Pathways
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Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) supplemented with cocktails of protease and phosphatase inhibitors (Sigma). A total of 20 µg protein was then separated by 8% SDS‑PAGE and then transferred onto a PVDF membranes according to methods described previously.23 (link),24 (link) Antibodies against DAB2IP (dilution 1:1000; #ab87811, abcam), cyclin D1 (dilution 1:1000; 26939-1-AP, proteintech), p21 (dilution 1:1000; 10355-1-AP, proteintech), p-AKT (Ser473) (dilution 1:1000; #4058, CST), AKT (dilution 1:1000; #9272, CST), p-ERK (dilution 1:1000; #4370, CST), ERK (dilution 1:1000; #4695, CST) and Tubulin (dilution 1:2000; #AF5012, Beyotime) were used in this study.
6
Ouabain-Induced Neuronal Apoptosis Evaluation
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Ouabain octahydrate (Cat #O3125) was purchased from Sigma Aldrich (USA) and was diluted to 10 mM in ultra-pure water and further used for cell treatment. Trypsin-EDTA, fetal bovine serum, penicillin-streptomycin, Hank’s Balanced Salt Solution, and trypan blue were purchased from Paneco (Russia). Neurobasal Medium, Supplement B-27, and 0.5 mM GlutaMax were purchased from Gibco (USA). MMLV RT Kit and TaqMan qPCR mix-HS were purchased from Evrogen (Russia). Primers were purchased from DNA-synthesis (Russia). GeneChip Rat Gene 2.0 ST Array; GeneChip WT PLUS Reagent Kit; GeneChipH WT Terminal Labeling Kit; and GeneChip Hybridization, Wash, and Stain Kit were purchased from Affymetrix (USA). Poly-L-ornithine, RIPA buffer, MgCl2, trichloroacetic acid , KCl, reagents for SDS-PAGE, cocktails of protease and phosphatase inhibitors, 3-(4,5-di methyl thiazol -2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and dimethyl sulfoxide were purchased from Sigma Aldrich. DC Protein Assay Kit was purchased from Bio-Rad (USA). Antibodies phospho-CREB (Ser133) (87G3) Rabbit mAb; β-Actin (D6A8) Rabbit mAb; Bcl-2 Rabbit pAb; Bax Rabbit pAb; and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology (USA). SuperSignal West Pico Chemiluminescent Substrate, SuperSignal West Femto Chemiluminescent Substrate, DNase I, and PureLink RNA Mini Kit, SYTO13 were purchased from Thermo Fisher Scientific (USA).
7
Evaluating MAP Kinase Phosphorylation
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Western blotting was used to evaluate the changes in the phosphorylation of the MAP kinases studied. The procedure was carried out in the same manner as described in our earlier work [42 (link)]. Cultured cells were lysed in RIPA buffer (Sigma) containing cocktails of protease and phosphatase inhibitors (Sigma). Then, the protein concentration was measured using DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Primary antibodies to the following proteins were used: p-p38 (cat.no #4511), and p38 (cat.no #9212) (Cell Signaling Technology, (CST)); p-JNK (cat.no sc-12882), JNK (cat.no sc-571), and β-actin (cat.no sc-47778) (Santa Cruz Biotechnology, (SCBT)); and secondary horseradish peroxidase conjugated antibodies (anti-rabbit, anti-mouse, and anti-goat) (SCBT, CST). Membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate or SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, MA, USA). Luminescence was detected by means of ChemiDoc XRS+ system (Bio-Rad), and the luminescence intensity was calculated with Image Lab 3.0 software (Bio-Rad).
8
Protein Quantification with BCA Assay
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Cocktails of protease and phosphatase inhibitors and the bicinchoninic acid assay (BCA) were purchased from Sigma (Sigma-Aldrich Company, St. Louis, MO, USA). Prestained protein standard was obtained from Bio-Rad (Bio-Rad, São Paulo, Brazil). All other reagents were of analytical grade and obtained from standard commercial suppliers.
9
Western Blot Analysis of ERK1/2 Activation
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ERK1/2 activation was evaluated using Western blotting. The procedure was carried out in the same manner as described in our earlier work [52 (link)]. Briefly, cultured cells in the 6-well plates were lysed in RIPA buffer (Sigma, Ronkonkoma, NY, USA) containing cocktails of protease and phosphatase inhibitors (Sigma, Ronkonkoma, NY, USA). The protein concentration was measured using DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Luminescence was detected by means of ChemiDoc XRS+ system (Bio-Rad, Hercules, CA, USA), and the luminescence intensity was calculated with Image Lab 3.0 software (Bio-Rad, Hercules, CA, USA).
10
Western Blot Analysis of Protein Targets
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Whole protein extracts were lysed in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing cocktails of protease and phosphatase inhibitors (Sigma, St. Louis, MO, USA) according to the manufacturer’s protocol. Total proteins from each lysate were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes and then blocked with 5% nonfat milk for 1 h. The membranes were then probed with the indicated primary antibodies at 4°C with gentle shaking overnight and incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies. Then, the proteins were visualized by chemiluminescence, and signals were quantified by Image J software. Antibodies used in this study are as follows: XEDAR (Bio-Rad, Hercules, CA, USA; AHP1182); LXRα (Abcam, Cambridge, UK; ab41902); RELA (Abcam, Cambridge, UK; ab16502); CD44 (Cell Signaling Technology (CST), Boston, MA, USA; 37259); CyclinD1 (CST, Boston, MA, USA; 2978); β-catenin (CST, Boston, MA, USA; 9562); EGFR (Abcam, Cambridge, UK; ab52894); FXOM1 (Abcam, Cambridge, UK; ab207298); N-cadherin (CST, Boston, MA, USA; 4061); E-cadherin (CST, Boston, MA, USA; 3195); Snail (CST, Boston, MA, USA; 3879); GAPDH (CST, Boston, MA, USA; 5174). The dilution factor for antibodies was 1:1,000.
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