For immunostaining or western blotting, primary antibodies against the following proteins were used: MYOF (Santacruz); EGFR, pEGFR, EPHA2, and EPHA2-pS897 (Cell Signaling); VIM and E-cadherin (Abcam); and actin (Pierce). For western blot analysis, cells were washed once with ice-cold PBS and then lysed in cold RIPA buffer supplemented with protease inhibitor cocktail (Roche Applied Biosciences). Cell lysate was centrifuged at 12,000 × g for 30 min, boiled in 2 × loading sample buffer, separated on a 10% gradient SDS–PAGE gel and blotted onto a PVDF membrane. The membrane was blocked with 0.1% Tween-20 in TBS containing 5% non-fat milk and probed with the indicated primary antibody, followed by incubation with the appropriate secondary antibody conjugated to horseradish peroxidase (Sigma), and the signals were visualized via ECL (Millipore).
Conjugated to horseradish peroxidase
Manufactured by Merck Group
Sourced in United States
Conjugated to horseradish peroxidase. A protein-enzyme complex commonly used in immunoassays and other applications that require signal amplification.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Epha2 ps897, by Cell Signaling Technology (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti h2b, by Abcam (1 mentions)
Luminata crescendo western hrp substrate, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Western blot substrate kit, by Bio-Rad (1 mentions)
Mouse anti actin, by Merck Group (1 mentions)
Laemmli sample buffer, by Merck Group (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Beclin 1, by Abcam (1 mentions)
Psd95, by Cell Signaling Technology (1 mentions)
7 protocols using conjugated to horseradish peroxidase
1
Immunoblotting and Immunostaining Assay
2
Western Blot Analysis of PrP2 in Embryos
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Five dechorionated and deyolked [47] (link) embryos were homogenized in extraction buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol) with protease and phosphatase inhibitors (Roche, France). Twenty microliters of homogenate were separated on 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. For antibody revelation, nitrocellulose membranes were incubated in Tris-buffered saline with 0.1% Tween 20 (TBST) supplemented with 5% skim milk powder for one hour and then soaked overnight at 4°C in TBST supplemented with 5% skim milk powder with the appropriate antibody, anti-PrP SAF84 (dilution 1∶1000) or anti-H2B (Abcam, UK, at 1∶1000) Immunosignals were visualized with anti-mouse or anti-rabbit IgG antibodies conjugated to horseradish peroxidase at 1∶2000 dilution (Sigma, France) and revealed with Luminata Crescendo Western HRP substrate (Millipore, France). Detection was done on the LICOR Odyssey and proteins bands were quantified with the NIH ImageJ sofware. At least three separate experiments were analysed and a ratio of PrP2 to H2B was then determined.
3
Drebrin Protein Expression in Chick DRG
4
Immunoblotting for Protein Analysis
5
Immunoblotting Analysis of Autophagy and Innate Immune Signaling
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Whole cell lysates were prepared in RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with a mixture of protease and phosphatase inhibitors and separated by SDS-PAGE for immunoblotting. Primary antibodies against Beclin1 (Cat. ab62557, 1:1500), Abcam, Cambridge, MA, USA), LC3B (Cat. 12741, 1:1000), Cell Signaling, Danvers, MA, USA), P62/SQSTM1 (Cat. ab91526 (1:2000), Abcam, Cambridge, MA, USA), ZIKV anti-E (Cat. GTX133314, 1:1000) Genetex, Irvine, CA, USA), TLR3 (Cat. sc-28999, 1:200), TLR4 (Cat sc-30002, 1:200), TLR5 (Cat. sc-10742, 1:200), MyD88 (Cat. sc-74532, 1:200), PERK (Cat. sc-377400, 1:200), IRE1α (Cat. sc-390960, 1:200), ATF6α (Cat. sc166659, 1:200), DDIT3 (Cat. sc-7351, 1:200) and β actin (Cat. sc-47778, 1:200) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were followed by incubation with a respective secondary antibody conjugated to horseradish peroxidase (Millipore, Billerica, MA, USA) used at specified dilution. The immunoblots were exposed to SuperSignal West Femto Substrate (Thermo Scientific, Waltham, MA, USA) and visualized using a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).
6
Hippocampal Protein Expression Analysis
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Protein concentration in the hippocampus was determined as in our previous study. A quantity of 20–40 μg of protein was loaded onto an 8–15% gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane and probed with the following primary antibodies: RIP1 antibody (1:1000, Abcam, United States); RIP3 antibody (1:1000, Abcam, United States); neuroligin (1:500, Abcam, United States); neurexin (1:500, Abcam, United States); PSD95 (1:1000, Cell Signaling); CREB (1:1000, Cell Signaling); phospho-CREB (p-CREB) (1:1000, Cell Signaling); BDNF (1:1000, Santa Cruz Biotechnology); β-actin (1:2000, Sigma-Aldrich) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to mouse anti-rabbit/mouse immunoglobulin G (1:10,000, Sigma-Aldrich).
7
Molecular Markers in Cell Signaling
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Protein concentration in the supernatants of cells or ipsilateral cortex was determined using a BCA protein assay kit (Pierce Biotechnology, Inc.). A quantity of 30–50 μg of total proteins was loaded onto a 10–15% gradient polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane and probed with the following primary antibodies: Bax antibody (1:1000, Santa Cruz Biotechnology, CA, USA), Bcl-2 antibody (1:1000, Santa Cruz Biotechnology), Cleaved Caspase-3 (1:500, Cell Signaling Tech. MA, USA), Caspase-3 (1:1000, Cell Signaling), Phospho-extracellular signal-regulated kinase (ERK)1/2 (1:2000, Cell Signaling), ERK1/2 (1:2000; Cell Signaling), Phospho-Akt (Ser 473) (1:500, Cell Signaling), Akt (1:1000; Cell Signaling), Brain-derived neurotrophic factor (BDNF, 1:1000, Santa Cruz Biotechnology), Vascular endothelial growth factor (VEGF, 1:100, Santa Cruz Biotechnology). β-actin (1:2000; Sigma-Aldrich) was used as an internal control. Secondary antibodies were horseradish peroxidase conjugated to goat/mouse anti-rabbit IgG (1:8000, Sigma-Aldrich). The membranes were developed using an enhanced chemiluminescence detection system (Pierce, Rockford, IL).
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