Mouse retinal tissues were dissected and pooled from six eyes, lysed in buffer (20 mM HEPES, pH 7.0, 10 mM KCl, 2 mM MgCl2, 0.5% Nonidet P-40, 1 mM Na3VO4, 1 mM PMSF, and 0.15U ml–1 aprotinin) and homogenized. Protein concentrations were determined using the Bradford colorimetric assay. Thirty micrograms of each protein lysate were loaded in each lane in sample buffer (2% SDS, 10% glycerol, 0.001% bromophenol blue, 1% DTT, and 0.05 M Tris-HCl, pH 6.8), separated on 10% SDS-PAGE (Invitrogen), and transferred to a PVDF membrane (Millipore, Temecula, CA). The blots were blocked with 5% nonfat milk in PBS and incubated with specific rabbit polyclonal antibody against ADAR2 and β-actin, followed by peroxidase-conjugated goat anti-rabbit IgG2a (Millipore) and the enhanced chemoluminescence detection system (Amersham Biosciences, Arlington Heights, IL).
Enhanced chemoluminescence detection system
Manufactured by Cytiva
Sourced in Germany
The Enhanced Chemoluminescence Detection System is a laboratory equipment designed to detect and analyze chemiluminescent signals. It provides a sensitive and quantitative method for the detection of proteins, nucleic acids, and other biomolecules using chemiluminescent techniques.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Biotrend (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti pp38, by New England Biolabs (1 mentions)
Anti akt, by New England Biolabs (1 mentions)
Anti erk, by New England Biolabs (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti ph2ax ser139 rabbit mab, by Cell Signaling Technology (1 mentions)
3 protocols using enhanced chemoluminescence detection system
1
Quantifying ADAR2 protein levels
2
Analysis of Cardiac Signaling Pathways
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Frozen muscle strips or pieces of the left ventricle were thawed on ice in 50 μL of homogenization buffer [1% (v/v) NP 40 (IGEPAL CA‐630), 10% (v/v) glycerol, 137 mM NaCl, 20 mM Tris–HCl pH 7,4, 20 mM NaF, 1 mM Na‐orthovanadate, 1 mM Na‐pyrophosphate, 50 mM β‐glycerophosphate, 10 mM ethylene‐diamine‐tetra‐acetic acid (EDTA) pH 8, 1 mM ethylene glycol‐bis‐(2‐aminoethyl)‐tetra‐acetic acid (EGTA) pH 7, 4 μg/mL aprotinin, 4 μg/mL leupeptin, 4 μg/mL pepstatin A, 1 mM phenylmethanesulfonyl fluoride (PMSF)] and homogenized. Protein of the suspensions was determined with BCA™ Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) and 20 μg of samples subjected to SDS‐PAGE. Western blotting was carried out according to standard protocols. The following antibodies were used: anti‐P‐AKT, anti‐AKT, anti‐P‐GSK3β, anti‐P‐p38, anti‐P‐ERK, anti‐ERK, anti‐P‐JNK (New England Biolabs, Ipswich, MA, USA), anti‐GAPDH (BioTrend, Cologne, Germany), anti‐mouse IgG, horseradish peroxidase‐linked, and anti‐rabbit IgG horseradish peroxidase‐linked (Amersham Biosciences, Freiburg).
For quantification, an enhanced chemo luminescence detection system (Amersham) was used according to the manufacturer's instructions.
For quantification, an enhanced chemo luminescence detection system (Amersham) was used according to the manufacturer's instructions.
3
Immunoblotting for pH2AX and IKBKB
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