Livers were lysed (150 mM NaCl, 20 mM Tris-HCl, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 10 mM NEM, PhosSTOP, cOmplete EDTA-free protease inhibitor cocktail) and protein quantified using Bradford Assay (Bio-Rad). Samples were electrophoresed on Mini PROTEAN TGX Precast Gels 4-15% (Bio-Rad) and transferred to 0.2-μm Protran nitrocellulose membranes. Membranes were blocked (5% skim milk powder in TBST, 50 mM Tris, 150 mM NaCl, pH 7.6, 0.1% Tween-20) for 1 hour at room temperature and then incubated with mouse monoclonal GSK6F05 anti-REVERBa, rabbit polyclonal anti-GR (Proteintech), mouse monoclonal anti-ACTB (Proteintech), or rabbit polyclonal anti-ONECUT1 (HNF6) (Proteintech) antibodies overnight at 4°C. Membranes were washed in TBST and secondary HRP-linked antibodies (GE Healthcare), or Dylight-680/800 secondary antibodies (Cell Signaling Technology) were incubated for 1 hour. After washes in TBST, immunoreactive bands were detected using SuperSignal West Dura (Thermo Fisher Scientific), and chemiluminescence was visualized on Kodak BioMax MR or XAR Film or LI-COR Odyssey CLx.
Dylight 680 800 secondary antibodies
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
DyLight-680/800 secondary antibodies are fluorescently-labeled antibodies that can be used to detect and visualize target proteins in various biological applications, such as Western blotting, immunohistochemistry, and flow cytometry. These antibodies are available in two different fluorescent dye options, DyLight 680 and DyLight 800, which emit at different wavelengths to accommodate different detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
Hrp linked antibodies, by GE Healthcare (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Complete mini protease inhibitor tablet, by Roche (1 mentions)
Nupage sample buffer, by Thermo Fisher Scientific (1 mentions)
Tandem mass tag reagent, by Thermo Fisher Scientific (1 mentions)
Odyssey blocking buffer tbs, by LI COR (1 mentions)
Gapdh sc 25778, by Santa Cruz Biotechnology (1 mentions)
2 protocols using dylight 680 800 secondary antibodies
1
Western Blot Analysis of Liver Proteins
2
Antibody sourcing for protein detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies against RASA3 (sc-166442 and sc-34468), PLEKHA1 (sc-50468), ADAP1 (sc-47836) and GAPDH (sc-25778) were from Santa Cruz Biotechnology (Insight Scientific, Middlesex, UK). The antibody against PHLDB1 (HPA038448) was from Sigma Aldrich (Poole, UK). The antibody against DAPP1 (AF7024) was from R&D Systems (Abingdon, UK). The antibody against GST (2622) and DyLight 680/800 secondary antibodies were from Cell Signaling Technologies (New England Biolabs, Hitchin, UK). Anti-rabbit HRP-coupled secondary antibody was from Jackson ImmunoResearch (Stratech Scientific Ltd, Ely, UK). Immobilon FL polyvinylidene difluoride (PVDF) was from Merck Millipore (Dorset, UK). Odyssey blocking buffer (TBS) was from LI-COR Biosciences (Cambridge, UK). Blank control beads, PtdIns(3,4)P 2 beads (P-B034A), free PtdIns(3,4)P 2 (P-3408) and PIP Arrays (P-6100) were from Echelon Biosciences, distributed via Tebu-Bio (Peterborough, UK). cOmplete mini protease inhibitor tablets were from Roche Life Sciences (Welwyn Garden City, UK). Tandem Mass Tag reagents and NuPAGE sample buffer were from Thermo Fisher Scientific (Loughborough, UK). Leukocyte removal filters were from Pall (Portsmouth, UK). All other reagents were from Sigma Aldrich (Poole, UK) unless otherwise stated in the relevant methods subsection.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!