A549 cells were seeded at 0.8x106 cells per 35mm dish and grown to confluency. Cells were infected for 1 h with AdEmpty or AdFAST at an MOI of 10. As positive control, cells were treated with 100 μM etoposide for 24 hr or 1 μM staurosporine for 6 hr. Whole cell lysates were collected at varying time post-infection with 2x Laemmli loading buffer (62.5mM Tris HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% β-mercaptoethanol). Samples were resolved by 15% SDS-PAGE and transferred onto a PVDF membrane (Millipore). Membranes were probed with rabbit anti-cleaved caspase-3 monoclonal antibody (1:1000, Cell Signaling #9664) or full-length caspase 3 (1:1000, Cell Signaling #9662) and 1:5000 goat anti-rabbit IgG conjugated to HRP (Bio-Rad #170–6515). Alpha-tubulin was used as a loading control (1:5000, CalBiochem #CP06, 1:10 000 goat anti-mouse IgG conjugated to HRP, Bio-Rad #170–6516). Blots were developed and densitometry was conducted as described above.
Goat anti rabbit igg conjugated to hrp
Manufactured by Bio-Rad
Sourced in Sweden, United States
Goat anti-rabbit IgG conjugated to HRP is a secondary antibody used in immunoassays. It binds to rabbit primary antibodies and is conjugated to horseradish peroxidase (HRP), an enzyme that can be used to detect and quantify target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Goat anti mouse igg conjugated to hrp, by Bio-Rad (1 mentions)
Ab58695, by Abcam (1 mentions)
Ab73895, by Abcam (1 mentions)
Nbp1 21502, by Novus Biologicals (1 mentions)
Ab8227, by Abcam (1 mentions)
Alphaeasefc, by Bio-Techne (1 mentions)
Pierce enhanced chemilumescent ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha tag monoclonal antibody, by Cell Signaling Technology (1 mentions)
Goat anti mouse antibody conjugated to horseradish peroxidase, by Bio-Rad (1 mentions)
β actin rabbit monoclonal antibody, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal gfp antibody, by Abcam (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
4 protocols using goat anti rabbit igg conjugated to hrp
1
Caspase-3 activation in A549 cells
2
Antibody-based Protein Expression Analysis
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All antibodies were commercially produced, tested, and recommended for the application. The primary capture antibodies in the ELISA analyses were rabbit anti-human ferroportin peptide (ab58695; Abcam, Cambridge, UK), rabbit anti-human IRP2 (LS-C80347, LifeSpan Biosciences, Seattle, WA, USA), rabbit anti-human HIF2 (ab73895; Abcam, Cambridge, UK). Mouse anti-human Immunoglobin G (IgG) conjugated with horseradish peroxidase (HRP; #55788; BD, Franklin Lakes, NJ, USA) was used as secondary capture antibody. The primary antibody used in the Western blot analyses was a rabbit anti-human ferroportin (NBP1-21502; Novus Biologicals, Littleton, MA, USA). A rabbit anti-human β-actin antibody (ab8227; Abcam, Cambridge, UK) was used as loading control. For Western blot analyses, the secondary antibody was goat anti-rabbit IgG conjugated to HRP (Bio-Rad, Sundbyberg, Sweden). Primary and secondary antibodies were used at 1 μg/mL.
3
Quantifying Adenoviral Protein Expression
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293 and A549 cells were seeded at 1x106 and 0.8x106 cells per 35mm dish, respectively. Next day, the cells were infected for 1 h at a multiplicity of infection (MOI) of 10 with AdEmpty or AdFAST-HA. Following a 48 h incubation, whole cell lysates were collected using the cell lysis buffer as described by Lieber et al. [27 (link)]. Samples were separated by 15% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The resulting membrane was probed with a mouse anti-HA tag monoclonal antibody (1:1000, Cell Signaling #2367) and goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (1:10 000, Bio-Rad #170–6516). To confirm Ad replication, the membrane was reprobed for Ad5 fibre (1:20 000 mouse anti-fibre monoclonal antibody, clone 4D2, Neomarkers). The membrane was also probed with antibody to α-tubulin to confirm equal loading (1:5000 rabbit anti-α-tubulin antibody, AbCam #ab15246, 1:5000 goat anti-rabbit IgG conjugated to HRP, Bio-Rad #170–6515). Blots were developed using the Pierce Enhanced Chemilumescent (ECL) Western Blotting Substrate (Thermo Scientific). Densitometry was performed using AlphaEaseFC (Alpha Innotech).
4
Western Blot Analysis of Viral Proteins
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Cells were lysed in RIPA lysis buffer [150 mM NaCl, 1% Non-ionic detergent, 1% Sodium deoxycholate, 0.1%SDS, (Biomax, Republic of Korea)]. About 20 μg of proteins per sample were separated by SDS-PAGE using a 10%polyacrylamide gels and transferred to nitrocellulose membranes (Cytiva, USA). Membranes were blocked with 10% (w/v) non-fat dry milk in TBST buffer [100 mM Tris (pH 7.6), 150 mM NaCl, 0.1% (w/v) Tween-20] for 1 h at room temperature. Membranes were incubated with a 1:1,000 dilution of rabbit polyclonal anti-Gag antibody, 1:500 dilution of rabbit polyclonal anti-Env antibody, 1:5,000 dilution of rabbit monoclonal GFP antibody (Abcam, UK), and 1:5,000 dilution of rabbit monoclonal β-actin antibody (Cell Signaling Technology, USA) at 4°C overnight. The membranes were then incubated with a 1:10,000 dilution of secondary antibody, goat anti-rabbit IgG conjugated to HRP (BioRad, USA), for 1 h at room temperature. The chemiluminescent signal was visualized by exposing the blots to a chemiluminescence imaging system, E-blot (e-BLOT Life Science, China). Rabbit polyclonal anti-Env antibody was designed to be specific for PFV Env-SU domain and was custom-produced by AbClon (Korea).
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