Primary hippocampal neurons were plated at 70,000 cells/well in 96-well plates, and maintained for 5d in neurobasal media before treatment with FR 180204, IFNγ, and/or staurosporine. After treatments, cells were fixed in 4% paraformaldehyde/4% sucrose in PBS for 30 min and washed 2x in PBS. Cells were blocked in a 1:1 mix of Odyssey Blocking Buffer (Licor) and PBS for 30 min and incubated in primary antibody (rabbit anti-MAP2, 1:500; Millipore) in Odyssey Blocking Buffer:PBS at 4°C overnight. Cells were washed 3x with PBS and incubated in secondary antibody (anti-rabbit IgG-800CW, 1:5000; Licor) and DRAQ5 far-red DNA stain (0.5 μM, Biostatus) for 1h at room temp. Plates were washed 3x with PBS and imaged on a Licor Odyssey Imager.
Odyssey blocking buffer
Manufactured by Merck Group
Sourced in United States
Odyssey blocking buffer is a liquid solution designed for use in immunodetection assays. It is formulated to block non-specific binding sites on membranes or slides, preventing unwanted interactions during the detection process.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (8 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Tween 20, by Merck Group (3 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Odyssey clx imaging system, by LI COR (2 mentions)
Intercept pbs blocking buffer, by LI COR (2 mentions)
Odyssey protein molecular weight marker, by LI COR (2 mentions)
Bis tris precast gel, by Bio-Rad (2 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Odyssey imager, by LI COR (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Irdye 800cw donkey anti mouse, by LI COR (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
Oxphos cocktail, by Thermo Fisher Scientific (1 mentions)
Ap1063, by Merck Group (1 mentions)
Ap1064, by Merck Group (1 mentions)
16 protocols using odyssey blocking buffer
1
Quantifying Neuronal Survival and Morphology
2
Western Blot Analysis of Skeletal Muscle Proteins
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Total protein was extracted from approximately 60 mg of frozen gastrocnemius tissue, as previously described [20 (link)]. Quantitation of total protein was conducted using a bicinchoninic acid (BCA) protein assay, with all samples normalised to 2 µg/µL for use in Western blotting procedures. Protein samples (6 per group) were loaded into 15-well 12% polyacrylamide gels followed by separation via electrophoresis at 120 V for approximately 2 h. Following electrophoresis, total protein loading for each lane on the membrane was assessed using RevertTM 700 Total Protein stain, as per the manufacturer’s instructions. After each membrane was scanned, the total protein stain was removed before membranes were blocked using Odyssey blocking buffer (Millipore, Burlington, MA, USA) for 1 h, then incubated with the following antibodies overnight: anti-glucose transporter 4 (GLUT4, 1:1000; Abcam, Cat. no. ab654) or protein kinase B (Pan-AKT, 1:1000; Cell Signalling, Cat. no. 4691S). Membranes were washed, followed by a one-hour incubation with Li-Cor secondary antibodies (1:10,000, IRDye 680 goat anti-rabbit). Finally, protein expression was visualised with the Li-Cor Odyssey CLX imaging system. Protein expression of GLUT4 and Pan-AKT was normalised to total protein content.
3
Synthetic Histone Peptide Blotting
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Synthetic H2B (residues 108–117) and H2BS112GlcNAc (residues 108–116 with S112GlcNAc) peptides (Abcam) were spotted on a nitrocellulose membrane, air-dried, and blocked with Odyssey blocking buffer (Licor). IRDye-800-coupled secondary antibodies and Odyssey imaging were used for visualization. For immunoblotting, proteins were separated by SDS-PAGE, blotted onto Immobilon-FL membranes (Millipore), and blocked with Odyssey blocking buffer. Proteins were visualized using IRDye-800- or IRDye-680-coupled antibodies (Licor) and by Odyssey imaging.
4
Quantification of Phosphorylated PDH Subunits
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In all, 1 × 106 AML cell lines were lysed in RIPA buffer and lysates were boiled at 100 °C r 10 min in Laemmli buffer after measuring protein concentration using Pierce BCA protein kit (Cat #23227). Equal amount of 20 μg was loaded to 4–15% mini Protean TGX precast gels for SDS-PAGE (Bio-rad Cat#4561085) and transferred with a Biorad Trans-blot Turbo transfer system to LF-PVDF membranes (Trans-blot Turbo RTA Transfer kit Cat#1704274). Membranes were blocked in Odyssey blocking buffer (Cat#927-40000) for 1 h and then incubated overnight at 4 °C with either of the following primary antibody mix; 1:1500 P-PDH Ser 232 (rabbit, Millipore AP1063), 1:1500 P-PDH Ser 300 (rabbit, Millipore, Calbiochem AP1064), 1:1500 P-PDH293 (Millipore, Calbiochem AP1062), 1:1000 PDH E1 alpha (mouse, Abcam, ab110330), 1:3000 Actin (mouse, Santa Cruz Biotechnology, SC-47778), 1:3000 Actin (rabbit, Cell Signaling Technology, 4970) or Oxphos cocktail 1:3000 (Thermo Fisher Scientific Cat#45-8199). Scanning of the membranes was performed with an Odyssey Clx scanner (Li-Cor Biosciences) after incubation with the following secondary antibodies (1:3000); Alexa Fluor 6 goat anti rabbit (Invitrogen, A21109) and IRDye 800CW donkey anti mouse (Li-Cor 926-32212).
5
Immunoblotting of Murine Embryonic Fibroblasts
6
Western Blot Analysis of Protein Samples
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All the fractions were diluted with 10× SDS–PAGE reducing sample buffer to a final concentration of 1× and denatured at 95 °C for 5 min. 50 μL of each sample was run along with eE2 marker and an Odyssey Protein Molecular Weight Marker (Li-Cor) (L) on 4–20% Bis-Tris precast gels (Bio-Rad). Proteins were then transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was blocked by Intercept (PBS) Blocking Buffer (Li-Cor) for 1 h at 37 °C followed by incubating the blot with a 1:500 dilution of a purified 8A6 mouse overnight at 4 °C. Primary antibody dilution was prepared in Odyssey Blocking Buffer in PBS with 0.05% Tween 20 (Sigma-Aldrich). The secondary antibody, IRDye 800CW Goat anti-Mouse IgG (Li-Cor, 926-32210), was used at a 1:10,000 dilution. The Western blot was scanned using Li-Cor Odyssey software (v.3.0).
7
Western Blot Analysis of UPB1 Expression
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Cell supernatants containing 5 μg protein were fractionated on NuPAGE® 4–12 % Bis-Tris Mini Gels (Life technologies) and transferred to nitrocellulose membranes. Membranes were blocked using Odyssey blocking buffer (LI-COR). Subsequently, blots were incubated for one hour with a 1:1000 dilution of rabbit anti-UPB1 (Anti-UPB1 AV42467-100UG, Sigma-Aldrich) and 1:5000 dilution of mouse anti-alpha-tubulin antibodies in blocking buffer (50 % Odyssey blocking buffer, 50 % PBS and 0.1 % Tween). Membranes were washed three times and then incubated for one hour with a 1:10,000 dilution of IRDye800 conjugated goat anti-rabbit and IRDye680 conjugated donkey anti-mouse (both LI-COR) secondary antibodies, in the same blocking buffer as used for primary antibodies, with 0.01 % SDS. Blots were scanned and band intensities analysed using the LI-COR Odyssey infrared imaging system.
8
Western Blot Protein Transfer and Imaging
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Using the Trans Blot Turbo (settings of 25 V and 1.3 A for 15 min; Bio-rad) proteins were transferred to a low-auto-fluorescence PVDF membrane (Bio-rad), blocked for 1 h at room temperature with Odyssey Blocking Buffer (Li-Cor; Lincoln, NE, USA), then incubated with primary antibody overnight at 4 °C in Odyssey Blocking Buffer with 0.1% Tween-20 (Sigma-Aldrich). Membranes were then probed with corresponding Li-Cor anti-mouse or anti-rabbit fluorescent secondary antibodies for one hour at room temperature at dilutions of 1/10,000 in Odyssey Blocking Buffer with 0.1% Tween-20 (Sigma-Aldrich) and 0.1% Tween. Imaging was conducted using the Li-Cor Odyssey Clx imaging system. Scans were performed at intensities that did not result in any saturated pixels.
9
Western Blot Analysis of Drosophila Heads
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For Western-blot analysis, ten male flies per genotype were frozen at -80°C, vortexed, and sieved to obtain samples of heads. Heads were homogenized in 50 μl Laemmli Sample Buffer (Bio-Rad) containing 2-mercaptoethanol. Samples were denatured for 5 minutes at 95°C and then centrifuged at maximum speed (17,000 g) for 5 minutes. Samples (20 μl) were loaded on a 4–20% gradient polyacrylamide gel (Bio-Rad), and proteins were separated at 150 V for 30 minutes. Proteins were transferred onto PVDF membrane (Millipore), and the membrane was blocked for one hour in Odyssey Blocking Buffer (LI-COR). Primary antibodies (rabbit anti-phospho-ERK, Cell Signaling Technology #9101, 1:1000; mouse anti-α-tubulin, Sigma-Aldrich #T9026, 1:5000) were diluted in Odyssey Blocking Buffer containing 0.2% Tween 20, and the membrane was incubated overnight in this solution. Membranes were then rinsed and incubated for 40 minutes with secondary antibodies conjugated to infrared fluorophores (anti-mouse IRDye 680RD and anti-rabbit 800CW, LI-COR, 1:10,000), and staining was imaged using an Odyssey Fc scanner (LI-COR).
10
Western Blot Analysis of Denatured Proteins
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Proteins and cell lysates were denatured by heating to 95°C for 5 min in LDS Sample Buffer (Thermo, NP0008) supplemented with 1X reducing agent (Thermo, NP0009), resolved using 4–12% Bis-Tris Protein Gels (Thermo, NP0329BOX) run in MES SDS Running Buffer (Thermo, NP0002), and transferred to nitrocellulose membranes (Thermo, IB23002) using the iBlot 2 (Thermo). Membranes were blocked with Odyssey Blocking Buffer in TBS (LI-COR, 927–50000) for 2 h at RT and incubated with primary antibodies in Odyssey Blocking Buffer + 0.2% Tween-20 (Sigma, P9416) overnight at 4°C. Membranes were washed three times with 1X TBST (G-Biosciences, R042) and incubated with secondary antibodies for 2 h at RT. After washing three times with 1X TBST, blots were imaged with the LI-COR Odyssey (LI-COR Biosciences, Lincoln, NE). Antibodies and reagents: GAPDH (Thermo, AM4300, 1:5000, 0.25 μg/mL), goat anti-rabbit (LI-COR, 926–32211, 1:15000), goat anti-mouse (LI-COR, 926–68020, 1:15000), Chameleon Duo Pre-Stained Protein Ladder (LI-COR, 928–6000).
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