Whole-cell lysates of RBMVEC and rat cerebral cortex were separated on Mini-PROTEAN TGX 4–20% gels (Bio-Rad, Hercules, CA) by SDS-PAGE followed by immunoblotting as previously described (Altmann et al., 2015 (link), Brailoiu et al., 2016 (link)). Proteins were transferred to an Odyssey nitrocellulose membrane (LI-COR Biosciences, Lincoln, NE). After incubation with blocking buffer, membranes were incubated overnight with primary antibody against GPR55 (rabbit polyclonal against the N-terminus of rat GPR55 (1:1000, cat # ADI-905-900; Enzo Life Sciences, Inc., Farmingdale, NY). An antibody against β-actin (mouse monoclonal, 1:10,000; cat # A5441, Sigma-Aldrich) was used to confirm equal protein loading. Membranes were washed with Tris-buffered saline-Tween 20 (TBST) and incubated with the secondary antibodies: IRDye 800CW conjugated goat anti-rabbit IgG (1:10,000, Cat # 926-32211, LI-COR) and IRDye 680 conjugated goat anti-mouse IgG (1:10,000, Cat # 926-32220, LI-COR) for 1 h at room temperature. Membranes were then washed in TBST and scanned using a LI-COR Odyssey Infrared Imager. Densitometric analysis was performed using Odyssey V.3 software (LI-COR).
Irdye 680 conjugated goat anti mouse igg
Manufactured by LI COR
Sourced in United States
The IRDye 680 conjugated goat anti-mouse IgG is a fluorescently labeled secondary antibody designed for use in various immunoassay applications. It specifically binds to mouse immunoglobulin G (IgG) molecules and can be detected using near-infrared fluorescence detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (5 mentions)
Irdye 800cw conjugated goat anti rabbit igg, by LI COR (2 mentions)
Mouse anti β actin mab, by Merck Group (2 mentions)
Irdye 800 conjugated goat anti rabbit igg, by LI COR (2 mentions)
Odyssey v3, by LI COR (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Mini protean tgx 4 20 gel, by Bio-Rad (1 mentions)
Odyssey nitrocellulose membrane, by LI COR (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Western lightning ecl, by PerkinElmer (1 mentions)
Hgt agarose, by Lonza (1 mentions)
5α dihydrotestosterone dht, by Merck Group (1 mentions)
Irdye800 conjugated anti rabbit igg, by Rockland Immunochemicals (1 mentions)
Mouse monoclonal anti human β tubulin, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mouse anti flag mab, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by HaiGene (1 mentions)
7 protocols using irdye 680 conjugated goat anti mouse igg
1
GPR55 Protein Expression Analysis
2
Western Blot Analysis of Protein Expression
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Western blot analysis was undertaken as described previously [33 (link)], with slight modifications. The samples were separated by SDS-PAGE under reducing conditions and blotted onto polyvinylidene difluoride membranes (Merck Millipore, Temecula, CA, USA). The membranes were incubated with the appropriate primary and secondary antibodies, washed, and visualized with an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). The mouse anti-Flag monoclonal antibody (mAb) was purchased from Abcam (Cambridge, UK). The rabbit anti-GPNMB polyclonal antibody and mouse anti-β-actin mAb were purchased from Sigma (Northbrook, IL, USA). The IRDye 680 conjugated goat anti-mouse IgG and the IRDye 800 conjugated goat anti-rabbit IgG were produced by Li-Cor Biosciences (Lincoln, NE, USA). The mouse anti-PRRSV nucleocapsid (N) protein mAb was produced and purified in our laboratory.
3
Western Blot Analysis of Viral Proteins
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The Western blot assay was carried out as previously described [36 (link)]. In brief, the lysates of PSV-infected PK15 cells and MBP-tagged recombinant VP1 protein were separated on SDS-PAGE gel and transferred to PVDF membranes. Membranes were incubated with either an mAb against VP1 protein (1:200), an anti-MBP mAb (1:10,000), or swine serum (1:100). Secondary antibodies were used with the IRDye 680 conjugated goat anti-mouse IgG (Li-Cor Biosciences, Lincoln, NE, USA) or goat anti-pig IgG (Biodragon-immunotech, Beijing, China) at a dilution of 1:10,000. Finally, membranes were scanned by an Odyssey infrared imaging system (Li-Cor Biosciences).
4
Western Blot Analysis of rVWF
5
Androgen Receptor Signaling Pathway Evaluation
6
Embryonic Protein Extraction and Western Blot
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Embryos were anesthetized with tricaine and mechanically homogenized with a Dounce homogenizer in NP40 buffer (2.5 µl per embryo) at 4°C. The protein was then spun down at 4°C for 20 minutes and the supernatant was aliquoted and frozen for storage at −80°C. Western blotting was performed using standard techniques with 4–20% Tris-glycine gel and transferred to PVDF membrane (all from Bio-Rad, Hercules, CA). Membranes were blocked with Odyssey blocking buffer, incubated in primary and secondary antibodies and imaged using the LI-COR Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Antibodies used were: polyclonal rabbit anti-Optineurin (C-term) (1∶1000, 100000; Cayman Chemical, Ann Arbor, MI), monoclonal mouse anti-gamma-tubulin (1∶1000, T6557, clone GTU-88; Sigma-Aldrich), IRDye800CW-conjugated goat anti-rabbit IgG and IRDye680-conjugated goat anti-mouse IgG (1∶1500, LI-COR Biosciences, Lincoln, NE). Western blots were performed at least three times with unique biological samples and yielded similar results. Each sample was a pool of 10 embryos from a single clutch.
7
Western Blot Analysis of SOCS3 and PRRSV
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Western blot assays were performed as previously described (59 ). In brief, cells were lysed by RIPA Lysis Buffer (Haigene, China) containing nuclease and protease inhibitors. Cell lysates (50 μg per well) were separated by 12% SDS-PAGE gel under reducing conditions and transferred to a PVDF membrane (Merck Millipore, USA). Membranes were incubated with the indicated primary antibodies and appropriate secondary antibodies. The mouse anti-SOCS3 mAb was purchased from Santa Cruz (sc-73045) and used at 1:200. The mouse anti-FLAG mAb (F-1804, Sigma) was diluted at 1:1000, and the mouse anti-β-actin mAb (A2228, Sigma) was used at 1:3000. The mouse anti-PRRSV nucleocapsid (N) protein mAb was produced and purified in our laboratory and was diluted at 1:10,000. The IRDye 680 conjugated goat anti mouse IgG was from Li-Cor Biosciences and used at 1:10,000. Finally, the membranes were scanned with Odyssey infrared imaging system (Li-Cor Biosciences, USA). The densitometric analysis was performed by Image J. The expression levels of indicated proteins were normalized by comparison with β-actin.
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