Cell and tissue extracts were prepared in lysis buffer (50 mmol/L Tris-HCl (pH = 8), 150 mmol/L NaCl, 0.2% SDS, 1% NP-40, 0.5% Deoxycholat, 1 mmol/L PMSF and protease- and phosphatase inhibitors (Roche Diagnostics, Manheim, Germany). Protein samples (10 μg per lane) were separated on 12.5% PAGE–SDS gel, transferred (Biometra, Göttingen, Germany) on nitrocellulose membranes (Whatman, Dassl, Germany), exposed to primary antibodies overnight at 4°C (ACADS, ACADM, ACADL, ACADVL, Sigma Aldrich, Steinheim, Germany; GAPDH, Ambion, Invitrogen, Karlsruhe, Germany) followed by exposure to fluorochrome-conjugated secondary antibodies (Li-COR Bioscience, Bad Homburg, Germany). For quantification of ACADS, ACADM, ACADL, ACADVL and GAPDH protein levels the Odyssey infrared imaging system (Li-COR Bioscience, Bad Homburg, Germany) was used.
Fluorochrome conjugated secondary antibody
Manufactured by LI COR
Sourced in United States
Fluorochrome-conjugated secondary antibodies are laboratory reagents used to detect and visualize target proteins in various biological assays. These antibodies are designed to bind to the primary antibodies that recognize specific proteins of interest, allowing for the detection and quantification of those proteins through fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (3 mentions)
Acadl, by Merck Group (1 mentions)
Gapdh, by LI COR (1 mentions)
Acadm, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Whatman, by Cytiva (1 mentions)
Rabbit anti phospho histone h2a x ser139, by Cell Signaling Technology (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Odyssey infrared detection system, by LI COR (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Ab214, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Image studio software, by LI COR (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Mouse anti ha, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
6 protocols using fluorochrome conjugated secondary antibody
1
Mitochondrial Enzyme Profiling in Cells
2
Western Blot Analysis of DNA Damage Markers
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Protein lysis buffer was prepared in 50 mmol/L HEPES, 100 mmol/L NaCl, 10 mmol/L EDTA, 1% Triton X-100, 4 mmol/L Na pyrophosphate, 2 mmol/L sodium orthovanadate, 10 nmol/L NaF, and 50 mmol/L B-glycerophosphate. Cells were lysed in lysis buffer containing a cocktail of proteinase inhibitors (Roche, Mannheim, Germany). Protein quantification of the lysates was performed using a BCA protein assay (Thermo Fisher, Waltham, MA, USA) and 30μg of protein was resolved on 4% to 20% polyacrylamide gels and transferred onto nitrocellulose membranes. The resulting membranes were incubated with blocking buffer (Li-cor Biosciences, Lincoln, NE, USA) and primary antibodies. The antibodies used were mouse polyclonal anti-MRE11 (ab214; abcam, Cambridge, UK), rabbit anti-phospho-histone H2A.X (Ser139) (#2577; Cell Signalling Technology, Danvers, MA, USA), and mouse monoclonal anti-β-actin (ab6276; abcam, Cambridge, UK). Fluorochrome-conjugated secondary antibodies (Li-cor Biosciences, Lincoln, NE, USA) were used and detected by infrared scanning densitometry using the Li-cor Odyssey Infrared Detection System (Li-cor Biosciences, Lincoln, NE, USA).
3
Western Blot Imaging and Quantification
4
Western Blot Analysis of Apoptosis Regulators
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After treatment, the cells were lysed and the proteins were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. After blocking non-specific binding sites with blocking buffer, the membranes were incubated overnight at 4 °C with primary antibodies (#6956 for NF-κB p65, #15071 for Bcl-2, #5023 for Bax and #2808 for survivin; Cell Signalling Tech, Beverly, MA, USA). β-actin (sc-47778, Santa Cruz Biotechnology Inc., Dallas, TX, USA) was used as a loading control. Following the removal of the primary antibodies, the membranes were washed three times with TBS (PBS containing 0.05% Tween 20) buffer at room temperature and later incubated with fluorochrome-conjugated secondary antibody (925–32211, Li-Cor Biotechnology, Lincoln, NE, USA). The membrane was then washed with TBS three times. Final detection was done with an Odyssey infra-red imaging system (Li-Cor Biotechnology).
5
Quantifying Viral Replication Inhibition
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The ICW assay was performed using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA) as previously described.41,42 (link) Briefly, A549 cells grown in 96-well plates (2 × 104 cells per well) infected with PR8, and treated or not with high concentrations (100, 150 and 200 μg mL−1) of selected compounds were fixed with 4% formaldehyde, washed, permeabilized with 0.1% Triton X-100 and incubated with Odyssey Blocking Buffer for 1 h (LI-COR Biosciences, Lincoln, NE, USA). The cells were then stained at 4 °C overnight with mouse anti HA (Santa Cruz Biotechnology, Germany) diluted in Odyssey Blocking Buffer. Cells were then washed and stained with a fluorochrome-conjugated secondary antibody (fluorescence emission at 800 nm) (LI-COR Biosciences, Lincoln, NE, USA) properly diluted in Odyssey Blocking Buffer. Subsequently, three washes with PBS plus 0.1% Tween 20 were performed and plates were analyzed by the Odyssey infrared imaging system (LI-COR). The relative fluorescence unit (RFU) was expressed as a percentage compared to untreated infected cells (100%). The 50% infectious dose (IC50) was defined as the dose of compound required to reduce viral replication by 50%. The Selectivity Index (SI) of each compound was calculated as the ratio between CC50 and IC50 (SI = CC50/IC50).
6
Protein Extraction and Quantification
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After being treated with IL-37 for 48h, cells were lysed by RIPA lysis buffer (Thermo Fisher) supplemented with 1% Protease Inhibitor cocktail (Roche). Then the lysis mixture was further used for total protein extraction. Total protein concentration was measured using a BCA protein assay kit (KeyGEN Biotec). Equal amounts of protein per sample were separated using 10% SDS-PAGE (YEASON, Shanghai, China) and transferred onto a polyvinylidene fluoride (PVDF) membrane, then blocking with 5% milk. The membrane was incubated overnight with the primary antibody (1:1000 or 1:2000) at 4°C, followed by fluorochrome-conjugated secondary antibody (1:10000, LI-COR Biosciences) for 1h at 37°C. The fluorescence intensity of the bands was measured using an Odyssey CLx Imaging System (Li-COR Biosciences).
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