Lysates (20 µg protein/lane) were separated by electrophoresis on SDS PAGE gels and transferred to polyvinylidene difluoride membranes, using Biorad Miniprotean system (BioRad). Blots were blocked and then incubated with antibodies against TP53 tumor suppressor protein (p53), B cell lymphoma 2 (BCL-2), and BCL-2 associated X protein (BAX) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), then further washed and incubated with corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology). Proteins were detected using Supersignal West FemtoChemiluminiscent substrate (Thermo Fisher Scientific, Rockford, IL, USA), and a Gel Doc Imaging system equipped with an XRS camera and Quantity One analysis software Image Lab 6.0 Biorad Laboratories). βactin (Sigma Aldrich) was used as a protein loading control.
Secondary peroxidase linked antibodies
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Secondary peroxidase-linked antibodies are laboratory reagents used in various immunoassays and detection methods. They function as a detection tool, providing a signal amplification system to enhance the sensitivity of the target analyte identification.
Automatically generated - may contain errors
Lab products found in correlation
Biorad miniprotean system, by Bio-Rad (3 mentions)
Supersignal west femto chemiluminiscent substrate, by Thermo Fisher Scientific (2 mentions)
Gel doc imaging system, by Bio-Rad (2 mentions)
Image lab 6, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Ecl detection reagent, by Cytiva (1 mentions)
Escitalopram oxalate, by Merck Group (1 mentions)
Edta na2, by Merck Group (1 mentions)
2 thiobarbituric acid, by Merck Group (1 mentions)
Bradford total protein concentration assay, by Bio-Rad (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Reduced glutathione, by Merck Group (1 mentions)
O phthalaldehyde, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Elisa immunoassay kit, by R&D Systems (1 mentions)
Supersignal west femto chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Quantity one analysis software, by Bio-Rad (1 mentions)
5 protocols using secondary peroxidase linked antibodies
1
Western Blot Analysis of Apoptosis Regulators
2
Western Blot Analysis of Cell Lysates
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Proteins in the culture medium of cell lysates were displayed in 12% SDS-PAGE and were electrophoretically transferred to immobile transfer membranes (Millipore, Bedford, MA). Transferred blots were incubated with a blocking solution containing 5% dry milk in TBST [0.15% Tween 20, 200 mmol/L NaCl, and 25 mmol/L Tris-HCl (pH 7.6)]. Blots were washed and incubated for 1 h with secondary peroxidase-linked antibodies (Santa Cruz, CA). Immunoreactive bands were detected using ECL detection reagents (Amersham, Buckinghamshir, UK) according to the manufacturer's instructions.
For the non-reducing condition, cell lysates were resuspended in lysis buffer without DTT. Protein samples were then mixed with SDS sample buffer without reducing agents (2% SDS, 10% glycerol, 0.01% bromophenol blue and 62.5 mM Tris–Cl pH 6.8), and the samples were not boiled prior to loading on a 10% SDS–PAGE.
For the non-reducing condition, cell lysates were resuspended in lysis buffer without DTT. Protein samples were then mixed with SDS sample buffer without reducing agents (2% SDS, 10% glycerol, 0.01% bromophenol blue and 62.5 mM Tris–Cl pH 6.8), and the samples were not boiled prior to loading on a 10% SDS–PAGE.
3
Biochemical Assays in Neuroscience
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Escitalopram oxalate, o-phthalaldehyde, reduced glutathione and Bradford reagent were from Sigma-Aldrich Chemicals GmbH (Munich, Germany), while EDTA-Na2 and 2-thiobarbituric acid were purchased from Merck KGaA (Darmstadt, Germany). Absolute ethanol, hydrogen peroxide and n-butanol were obtained from Chimopar (Bucharest, Romania). Antibodies against BDNF, MeCP2, β actin and secondary peroxidase-linked antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while phosphorylated histone. ELISA tests for caspase-3 were purchased from Elabscience (Houston, TX, USA) and Bradford total protein concentration assay was from BioRad (Hercules, CA, USA).
4
Immunoblot Analysis of Apoptosis Markers
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Lysates (20 µg protein/lane) underwent electrophoresis on SDS PAGE gels, followed by transfer to polyvinylidene difluoride membranes using the Biorad Miniprotean system (Bio-Rad Laboratories, Hercules, CA, USA). The blots were blocked and then incubated with antibodies against p53, BCL-2, BAX, COX2, NFκB, pNFκB, and NRF2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), as well as caspase-3 and caspase-9 (Antibodies Online, Atlanta, GA, USA). Subsequently, the blots were washed and exposed to corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein detection was performed using Supersignal West FemtoChemiluminiscent substrate (Thermo Fisher Scientific, Rockford, IL, USA), and analysis was conducted using a Gel Doc Imaging system equipped with a XRS camera and Quantity One® 1-D analysis software 4.6 (Bio-Rad Laboratories, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA) served as a protein-loading control.
5
Evaluation of Inflammatory Mediators
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Inflammation was assessed by the measurement of IL-31, IL-33, IL-6, TNF-α, and STAT3 levels using ELISA immunoassay kits from R&D Systems Inc. (Minneapolis, MN, USA). In addition, the expressions of transcription factor NFκB and its phosphorylated form (pNFκB) were evaluated by western blot analysis. For western blot, a 20 μg protein/lane was separated by electrophoresis on SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Bio-Rad Mini-PROTEAN System from Bio-Rad) as previously described [46 (link)]. Blots were blocked and then incubated with antibodies against NFκB, pNFκB p65 (Ser536) (93H1), and GAPDH and then further washed and incubated with corresponding secondary peroxidase-linked antibodies (Santa Cruz Biotechnology Inc.). The proteins were detected using the SuperSignal West Femto chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA) and were then quantified using Quantity One Analysis Software (Bio-Rad).
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