Cells were lysed in 25 mM HEPES (pH 7.4), 150 mM NaCl, 10% glycerol, and 1% NP40 and virions were dissolved after pelleting in 2xSDS sample buffer [100 mM Tris-HCl (pH 6.8), 4% SDS, 12% 2-mercaptethanol, 20% glycerol, 0.05% bromophenol blue]. Proteins in cell and viral lysates were fractionated by SDS PAGE, transferred to PVDF membrane (Millipore), and blocked with 4% milk in PBS containing 0.1% Tween 20. In all experiments, viral lysates were normalized by p24 immunoblot and/or ELISA levels and re-analyzed through additional rounds of immunoblotting. Subsequently, membranes were incubated with primary antibodies, horseradish peroxidase (HRP)-conjugated secondary antibodies, developed using HyGlo chemiluminescent HRP detection reagent (Denville Scientific) and exposed to film. Band intensity was analyzed by using Image J software.
Hyglo chemiluminescent hrp detection reagent
Manufactured by Thomas Scientific
HyGLO™ Chemiluminescent HRP Detection Reagent is a laboratory reagent used to detect the presence of horseradish peroxidase (HRP) enzyme in biological samples. It produces a chemiluminescent signal in the presence of HRP, allowing for sensitive detection and quantification of target proteins or molecules.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Ab129068, by Abcam (2 mentions)
Mab3574, by Merck Group (2 mentions)
Image lab 6, by Bio-Rad (2 mentions)
Ab201060, by Merck Group (2 mentions)
Nupage lds sample loading buffer, by Thermo Fisher Scientific (2 mentions)
660 nm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
4 protocols using hyglo chemiluminescent hrp detection reagent
1
Protein Characterization of Viral Lysates
2
Protein Extraction and Western Blot
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Cells were rinsed with cold PBS and harvested in 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 5 mM Na3VO4, 200 μM NaF, 21 μM leupeptin, 230 nM aprotinin, and 1 mM PMSF. Cell lysate was centrifuged at 10,000 × g for 10 minutes at 4°C. Protein concentration of the resulting supernatant was determined using a 660 nm Protein Assay kit (Thermo Scientific). Total cell lysate (30 μg) was boiled for 5 minutes and resolved in acrylamide/bisacrylamide gel by electrophoresis. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) or nitrocellulose membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% milk in PBST or TBST and incubated with primary and secondary antibodies according to manufacturer’s recommendations. Reactive bands were visualized by exposure to film using HyGLO Chemiluminescent HRP Detection Reagent (Denville Scientific, Metuchen, NJ) or SuperSignal West Dura Extended Duration Substrate (Thermo Scientific). Blots were stripped in 0.2 M NaOH with shaking for 10 minutes at room temperature.
3
Immunoblotting of Brain Protein Markers
4
Immunoblotting of Brain Protein Markers
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The cortex from E4FAD mouse brains was homogenized in Eppendorf tubes (kept on ice), using 300 ul of RIPA buffer (Thermo Fischer Scientific) with freshly added protease and phosphatase inhibitor cocktails. The samples were placed on an orbital shaker at 4°C overnight and spun the next day at 12000 rpm for 20 min at 4°C. The supernatant from each sample was collected and the protein concentration was assayed using BSA as working standard. Equal amounts of protein (40 μg) were heat-denaturized in NuPAGE LDS sample- loading buffer (Invitrogen) for 5 min at 95°C, resolved by SDS-PAGE and transferred to PVDF membranes (Sigma-Aldrich, MO, USA). The membranes were blocked with Tris-buffered saline (TBS) containing 0.05% Tween and 5% non-fat dry milk (NOX2, Aβ42) and Tris-buffered saline (TBS) containing 0.05% Tween and 2% BSA (Vinculin) and then incubated overnight with antibodies directed against NOX2 (Rabbit monoclonal, 1:5000, Abcam, ab129068), Aβ42 (Rabbit monoclonal, 1:500, Abcam, ab201060) or Vinculin (Mouse monoclonal, 1:1000, Millipore, MAB3574). Peroxidase conjugated IgG was used as secondary antibody. Membrane-bound immune complexes were detected by HyGLO™ Chemiluminescent HRP Detection Reagent (Denville Scientific). Protein loading was normalized according to Vinculin expression. Quantification was performed by densitometric analysis using Imagelab 6.0 software (Bio-Rad).
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