Cells (1 × 105 cells) were seeded in 2 mL growth medium in a 3-cm dish. Following a 24-h incubation period at 37 ˚C, cells were treated with vehicle (control), gluRDVs at the proportional concentrations to Dox-gluRDVs, free Dox, and Dox-gluRDVs at 0.3 μM Dox in growth media for 24 h, lysed using RIPA lysis buffer, centrifuged to collect the supernatants, and the protein concentration was determined by Bradford Protein Assay. The protein samples were separated by electrophoresis in a 10% polyacrylamide gel and transferred to PVDF membrane. The membranes were incubated with TBS plus 0.1% Tween 20 (TBST) and 5% bovine serum albumin (BSA) for 1 h at room temperature. After incubation, primary antibodies (ERK, dilution 1:500, Santa Cruz; p-ERK, dilution 1:500, Santa Cruz; Procaspase-3, dilution 1:1000, Cell signaling; Cleaved caspase-3, dilution 1:500, Cell signaling; GAPDH, dilution 1:10000, Santa Cruz) were added to TBST containing 1% BSA and incubated with the membranes at 4 ˚C overnight. The membranes were then incubated with HRP-conjugated secondary antibodies (dilution 1:5000, Santa Cruz) and were developed using the Luminata Cresendo Western HRP substrate (Millipore).
Luminata cresendo western hrp substrate
Manufactured by Merck Group
Luminata Crescendo Western HRP Substrate is a chemiluminescent detection reagent used for western blot analysis. It is designed to detect horseradish peroxidase (HRP)-conjugated secondary antibodies and provides a sensitive and stable luminescent signal.
Automatically generated - may contain errors
Lab products found in correlation
Procaspase 3, by Cell Signaling Technology (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Amersham hyperfilm ecl film, by GE Healthcare (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Cedarlane (1 mentions)
2 protocols using luminata cresendo western hrp substrate
1
Investigating Dox-Induced Cell Signaling and Apoptosis
2
Viral Infection Grading and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Following six weeks of urine co-culture, the viral infection was graded in a blind fashion according to CMV CPE with a scale of: 1+ (0–20% CPE), 2+ (20–50% CPE), 3+ (50–80% CPE), 4+ (80–100% CPE), and the urine co-cultures were harvested. The cultures were pelleted by centrifugation at 15,300 x g for 10 minutes, washed with PBS and pelleted again. The pellet was resuspended in protease inhibitor cocktail (Sigma) and RIPA buffer (50 mM Tris HCl pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA; adjusted to pH 7.4), and incubated on ice for 30 minutes. The lysate was centrifuged at 14,000 x g for 20 minutes at 4°C and the protein was quantitated using a Bio-Rad protein assay kit (Bio-Rad). Primary antibodies for western blotting included mouse anti-GFP (clone: BV-F4, 1:1,000, BioVision), mouse anti-RhCMV IE-1 (clone: 2A1.2, 1:750) kindly provided by Drs. Scott Hansen and Louis Picker (Oregon Health & Science University), and mouse anti-β actin (clone: AC15, 1:1,000; Sigma) as a lysate control. After probing with goat anti-mouse IgG (1:10,000, Cedarlane), the membrane was treated with Luminata Cresendo Western HRP substrate (Millipore) and exposed on an Amersham Hyperfilm ECL film (GE Healthcare).
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