The total protein concentration in cells and tissues was determined using bicinchoninic acid assay (BCA) (Beyotime, China) according to manufacturer’s instructions. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the detected proteins were transferred onto nitrocellulose filter (NC) membranes. Subsequently, the membranes were blocked in 5% skim milk for 1 h at room temperature with gentle shaking. After that, the membranes were incubated with primary antibodies of rabbit anti-CX3CR1 (1:500, Abcam, Cambridge), mouse anti-bcl-2 (1:500, Santa Cruz, CA, United States), mouse anti-caspase-3 (1:500, Santa Cruz, CA, United States), and/or mouse anti-β-actin antibody (1:1000, Proteintech, China) at 4°C overnight, followed by 1 h incubation with anti-rabbit IgG (H+L) (DyLight 800 Conjugate; Cell Signaling Technology, United States) or anti-mouse IgG (H+L) (DyLight 800 Conjugate; Cell Signaling Technology, United States) secondary antibody at room temperature. The protein bands were visualized with Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, United States), and the intensity of each band was analyzed using Image J (National Institutes of Health, United States).
Dylight 800 conjugate
Manufactured by Cell Signaling Technology
Sourced in United States
The DyLight 800 Conjugate is a fluorescent dye that can be used for protein labeling and detection in various applications. It has an excitation maximum at 783 nm and an emission maximum at 810 nm, making it suitable for near-infrared fluorescence imaging.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (4 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
H9658, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
α tubulin t6199, by Merck Group (1 mentions)
Ripa buffer, by Wuhan Servicebio Technology (1 mentions)
Rabbit anti vegfa, by Abcam (1 mentions)
Rabbit anti ang 1, by Abcam (1 mentions)
Rabbit anti ang 2, by Abcam (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Nitrocellulose nc membrane, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Phenylmethylsulfonyl fluoride, by Wuhan Servicebio Technology (1 mentions)
Proteinase inhibitor cocktail, by Wuhan Servicebio Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Gata2, by Proteintech (1 mentions)
Mouse anti caspase 3, by Santa Cruz Biotechnology (1 mentions)
Mouse anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Mouse anti β actin antibody, by Proteintech (1 mentions)
6 protocols using dylight 800 conjugate
1
Protein Expression Analysis by Western Blot
2
Quantifying HA-hERG Channel Expression
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This assay was used to quantify HA-hERG channel expression at the plasma membrane (PM) and full experimental details can be found in (Al-Moubarak et al., 2020 (link)). In brief, 48 hours after transfection, cells were incubated with primary antibody (mouse monoclonal anti-HA antibody (H9658, Merck Life Science United Kingdom Limited, Dorset)) diluted 1:1,000 at 4°C for 1 h. After washing (at 4°C), cells were fixed in 3.7% formaldehyde (252549, Merck Life Science United Kingdom Limited, Dorset) for 20 min at room temperature (RT). After fixation and washing, cells were incubated with Wheat Germ Agglutinin (WGA) 680 Alexa Fluor (W32465, Life technologies, Paisley, United Kingdom) at 5 μg/ml in Hanks’ Balanced Salt Solution (HBSS) for 10 min. After washing, a secondary antibody (anti-mouse IgG (H + L) (DyLight 800 conjugate) (5257S, Cell Signaling Technology, Leiden); 1:1,000 dilution) was added for 1 h. After three final washes, assay plates were imaged using a LI-COR® Odyssey CLx imaging system.
3
Western Blot Antibody Validation
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Chicken anti LDL-R (GW22458A, dilution: 1/500) and α-Tubulin (T6199, dilution 1/1000) were purchased from Sigma. Anti G (8G5F11, dilution 1/1000) was purchased from Kerafast. Goat anti-mouse Alexa fluor 488, purchased from ThermoFisher (A-11029, dilution 1/1000), were used as secondary antibodies. Goat anti-rabbit DyLight 800 conjugate (from Cell Signaling Technology, ref 5251, dilution 1/1000) was also used as a secondary antibody in western blot.
4
Quantifying Angiogenic Factors in Ischemic Brain
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The mice were euthanized under deep anesthesia using pentobarbital sodium (50 mg/kg, i.p.). The brain tissues in the ischemic area (about 50 mg) were homogenized in radioimmunoprecipitation assay (RIPA) buffer (Servicebio, China) containing 1% phenylmethanesulfonyl fluoride (PMSF). Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Equal amounts of proteins were separated on 10% SDS-polyacrylamide gels, and then transferred onto nitrocellulose (NC) membranes (Millipore, United States). Next, the membranes were blocked with 5% skim milk for 1 h at room temperature, and then incubated with primary antibodies of rabbit anti-VEGFA (1:1000; Abcam, United Kingdom), rabbit anti-Ang-1 (1:10000; Abcam, United Kingdom), rabbit anti-Ang-2 (1:1000; Abcam, United Kingdom) or rabbit anti-β-actin (1:1000; Cell Signaling Technology, United States) at 4°C overnight. After washing three times with Tris buffered saline Tween (TBST), the membranes were incubated with secondary antibody of anti-rabbit IgG (H + L) (DyLight 800 Conjugate) (1:10000; Cell Signaling Technology, United States) for 1 h at room temperature. The protein bands were observed with Odyssey Infrared Imaging System (Li-COR Biosciences, Lincoln, NE, United States), and the intensity of each band was analyzed using Image J software.
5
Western Blot Analysis of Protein Expression
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RIPA lysis buffer (Sigma, St. Louis, MO, USA) containing a proteinase inhibitor cocktail (Servicebio, Wuhan, China) and phenylmethylsulfonyl fluoride (Servicebio) was used to extract total protein in tissues. Samples with the same protein concentration were resolved on a 12% polyacrylamide gel and transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA). Membranes were blocked with protein-free rapid sealing solution (Servicebio) at room temperature for 15 min, incubated with the following antibodies at 4 ℃ overnight: GATA2 (1:1,000, Proteintech Cat# 11103-1-AP, RRID:AB_10914503), TFAP2A (1:1,000, Affinity Biosciences Cat# AF0535, RRID:AB_2834124), LMBRD1 (1:1,000, ABclonal Cat# A15866, RRID:AB_2763294), KRT8 (1:2,000, Proteintech Cat# 27105-1-AP, RRID:AB_2918117). The membranes were washed with Tris-buffered saline (100 mM NaCl, 50 mM Tris-HCl, pH 7.6) containing 0.1% Tween 20 thrice for 10 min each, probed with anti-rabbit immunoglobulin G (heavy + light) [IgG (H + L)] (DyLight 800 Conjugate) (1:30,000, Cell Signaling Technology Cat# 5151, RRID:AB_10697505) for 2 h at room temperature, and washed again. The membranes were scanned and imaged using the Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA). The intensity of each band was quantitatively determined by the ImageJ analysis system (Wayne Rasband, National Institutes of Health, USA).
6
Western Blot Quantification of Protein Phosphorylation
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Protein was extracted from ∼30 mg snap‐frozen tissue as previously described (Mallinson et al. 2012 ). Total and phosphorylated 4EBP1 (Ser65) and p70S6k (Thr389) (Cell Signaling, Danvers, MA, USA) and PDK4 (Millipore, UK) were analysed with anti‐mouse IgG (H+L) (DyLight 800 Conjugate, Cell Signaling) or anti‐rabbit IgG (H+L) (DyLight 680 Conjugate, Cell Signaling) as the secondary antibodies. Blots were scanned and bands identified using the Odyssey Infrared Imaging System (LI‐COR Biosciences, Lincoln, NE, USA). Dual probing was used for total and phosphorylated proteins. Density volume was adjusted by subtracting the local background and then normalised with β‐actin (Sigma Aldrich, Dorset, UK).
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