Odyssey blocking buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

Odyssey blocking buffer is a protein-free, phosphate-buffered saline solution designed for use in Western blotting and other protein detection applications. It is formulated to effectively block non-specific binding, helping to improve the signal-to-noise ratio in your assays.

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20 protocols using odyssey blocking buffer

Hippocampus Protein Extraction and Western Blotting

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Under deep anesthesia, animals were transcardially perfused with ice-cold PBS. After decapitation, the brain was removed, and the hippocampus total protein extraction was performed with RIPA buffer. Microglia cells were isolated with MACS from hippocampus samples. Total protein concentration of hippocampi and microglia samples w determined by using a BCA assay (Pierce). Proteins were denatured in Laemmli buffer at 95 °C for 5 min, and 20 μg of protein per sample was loaded into a 10% SDS gel. Following SDS-PAGE, proteins were transferred onto PVDF membrane (Immobilon-FL) activated in methanol at 100 V for 2 h by wet transfer. The membrane was blocked in Odyssey blocking buffer (LiCor Biosciences) for 3 h and incubated with the primary antibody (Abcam rabbit monoclonal anti-NLGN4:1/500, mouse anti-GAPDH:1/2500 in Odyssey blocking buffer and 0.2% Tween 20) overnight at 4 °C.Following three washing steps in TBS-T, the membrane was incubated with a secondary antibody (Invitrogen; 1/2500 goat IRDye800 anti-mouse, 1/2500 goat IRDye680 anti-rabbit in Odyssey blocking buffer, 0.2% Tween20 and 0.01% SDS) for 3 h at RT with slow shaking. The membrane was washed three times in TBS-T and proteins were detected by using Odyssey Imaging Systems (LiCor Biosciences).

Guneykaya D., Ugursu B., Logiacco F., Popp O., Feiks M.A., Meyer N., Wendt S., Semtner M., Cherif F., Gauthier C., Madore C., Yin Z., Çınar Ö., Arslan T., Gerevich Z., Mertins P., Butovsky O., Kettenmann H, & Wolf S.A. (2023). Sex-specific microglia state in the Neuroligin-4 knock-out mouse model of autism spectrum disorder. Brain, behavior, and immunity, 111, 61-75.

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LPS and KLH Stimulation of THP-1 Cells

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THP-1 cells (1 × 10⁷ cells) were stimulated with 0.1 µg/ml LPS or 500 µg/ml KLH at 37° C for various periods. At the end of the incubation period, cells were lysed in a RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM disodium ethylene-diaminotetraacetate [Na₂EDTA], 1 mM ethylene glycol tetraacetic acid [EGTA], 1 % Nonidet P-40 [NP-40], 1 % sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 µg/ml leupeptin, Protease Inhibitor Cocktail [Nakarai Tesque, Tokyo], 1 mM NaF and 1 mM phenylmethylsulfonyl fluoride [PMSF]) for 30 min on ice. After centrifugation (20,000 g, 10 min, 4° C), supernatant protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad Laboratories, CA), blocked with an Odyssey Blocking Buffer (IL-COR, Lincoln, NE). The membrane was incubated with the appropriate antibodies 1:600 (vol/vol) in the Odyssey Blocking Buffer with 0.2 % (vol/vol) Tween 20, followed by an incubation with a goat anti-rabbit IgG Alexa Fluor 680 secondary antibodies (Thermo Fisher Scientific, diluted at 3:10,000) in the Odyssey Blocking Buffer. The stained protein bands were digitally detected in an Odyssey Fc Dual-Mode Imaging System (IL-COR, Lincoln, NE).

Yasuda K, & Ushio H. (2016). Keyhole limpet hemocyanin induces innate immunity via Syk and Erk phosphorylation. EXCLI Journal, 15, 474-481.

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Western Blot Analysis of Protein Extracts

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Protein concentrations of whole cell lysates were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Lysates of equal protein concentrations were prepared in LDS sample buffer (Invitrogen, Carlsbad, CA, USA), separated on denaturing NuPAGE 4-12% polyacrylamide gradient gels (Invitrogen), and transferred to nitrocellulose membranes (GE Healthcare, Amersham, UK). Membranes were blocked in a 1:1 mixture of Odyssey blocking buffer (Li-Cor, Lincoln, NE, USA) and Tris-buffered saline (TBS). Membranes were then incubated with primary antibodies in a 1:1 mixture of Odyssey blocking buffer and TBS containing 0.1% Tween 20 (Fisher BioReagents, Waltham, MA, USA) overnight at 4°C. Following washing with TBS with 0.1% Tween 20, membranes were incubated with IRDye secondary antibodies (Li-Cor) for 1 h at room temperature. Membranes were then washed with TBS and analyzed using the Odyssey CLx infrared imaging system (Li-Cor) according to the manufacturer's instructions.

Kocab A.J., Veloso A., Paulsen M.T., Ljungman M, & Duckett C.S. (2015). Effects of Physiological and Synthetic IAP Antagonism on c-IAP-Dependent Signaling. Oncogene, 34(43), 5472-5481.

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HeLa Cell Protein Extraction and Western Blot

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HeLa cells were centrifuged and washed once with cold PBS before preparation of extract or freezing the pellets in liquid nitrogen. For the preparation of extracts, cells were lysed in 20 mM Tris-HCl (pH 7.7), 100 mM KCl, 50 mM sucrose, 1 mM MgCl₂, 0.1 mM CaCl₂, 0.5% TX-100 (APC-buffer) containing protease inhibitor cocktail (Roche, 04693132001) and phosphatase inhibitor PhosSTOP (Roche, 4906837001) for 7 min on ice, and cell lysates were cleared by centrifugation. Equal amounts of samples were loaded and run on 4–20% gradient gels followed by semi-dry transfer with Trans-blot (Bio-Rad, Hercules, CA). Membranes were blocked in 5% milk or Odyssey blocking buffer (Fisher Scientific, NC9877369) in TBS for 1 h, followed by primary antibody incubation for 1 h at RT or overnight at +4°C in TBST (TBS containing 0.05% Tween), and secondary antibody for 1 h at RT in TBST. Primary antibodies against Hec1 (Abcam, ab3613), cleaved PARP (Cell Signaling, 9546, dilution) and GAPDH (Advanced ImmunoChemical Inc., mAb 6C5) were followed by secondary antibody Alexa Fluor^® anti-mouse 680 (Invitrogen). Signals were detected using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

Laine L.J., Mäki-Jouppila J.H., Kutvonen E., Tiikkainen P., Nyholm T.K., Tien J.F., Umbreit N.T., Härmä V., Kallio L., Davis T.N., Asbury C.L., Poso A., Gorbsky G.J, & Kallio M.J. (2021). VTT-006, an anti-mitotic compound, binds to the Ndc80 complex and suppresses cancer cell growth in vitro. Oncoscience, 8, 134-153.

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Western Blot Analysis of Protein Extracts

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Kocab A.J., Veloso A., Paulsen M.T., Ljungman M, & Duckett C.S. (2015). Effects of Physiological and Synthetic IAP Antagonism on c-IAP-Dependent Signaling. Oncogene, 34(43), 5472-5481.

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Protein Extraction and Western Blot Analysis

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HEK293 cells were lysed on ice for 60 min in lysis buffer containing 10 mM Tris (pH 7.4), 1% Triton X-100, 150 mM NaCl, 10% glycerol, and freshly added protease inhibitors (Roche Complete Protease Mini, Cat # 4693159001) and phosphatase inhibitors (PhosStop pellets, Sigma Aldrich, Cat # 4906845001). Cell lysates were centrifuged at 15,000 × g for 20 min at 4°C. Supernatant was collected as the soluble fraction. The pellet was washed 3 times with lysis buffer and centrifuged at 15,000 × g for 5 min each at 4°C. The pellet was resuspended in lysis buffer supplemented with 4% SDS, sonicated 3 times, boiled for 30 min, and collected as the insoluble fraction. Protein concentration was determined using Lowry Protein Assay (Bio-Rad). For Western blot analysis, proteins were subjected to 4%–12% bis-tris midi gels (Bio-Rad) and transferred to nitrocellulose membrane. Membrane was blocked with Odyssey Blocking Buffer (Fisher Scientific). The following primary antibodies were used: mouse anti-Myc (Cell Signaling, Cat # 2276, 1:1000 dilution) and mouse anti-beta tubulin (Sigma-Aldrich, Cat # T8328, 1:8000 dilution). Appropriate IRDye infrared secondary antibodies were purchased from LI-COR Biosciences and used at a dilution of 1:10000. Odyssey Infrared Imaging System (LI-COR Biosciences) was used to detect the signal of target proteins.

Xu H., An J.J, & Xu B. (2017). Distinct cellular toxicity of two mutant huntingtin mRNA variants due to translation regulation. PLoS ONE, 12(5), e0177610.

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Quantifying HA-tagged Protein Expression

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Stably transfected HEK293 cell lines grown to approximately 90% confluency in 6-well dishes were chilled on ice for 5 min. Following a wash with ice-cold 1x Phosphate-buffered saline PBS (137 nM NaCl, 10 mM NaH₂PO₄, 2.7 mM KCl, pH 7.4), cells were covered with 200 μl lysis buffer (100 mM Tris (pH 7.4), 150 mM NaCl, 0.5% CHAPS, 1 mM EDTA, 6 mM MgCl₂ and 100 mM PMSF) and incubated on ice for 5 minutes. Cells were then scraped and lysates were sonicated and spun down at 10,000 × g and 4°C. The supernatant was collected and protein concentration was determined by the method of Bradford (Bradford 1976 (link)). The samples were normalized to total protein, and 25 μg protein of each sample was run on a 10% Tris-glycine SDS-PAGE. The separated proteins were transferred to nitrocellulose and immunoblotting was performed using a mouse monoclonal anti-HA11 antibody and anti-GAPDH antibody (Cat# 649201, Biolegend, San Diego, CA, USA: RRID:AB_2107422). Both primary antibody was diluted 1:1000 in Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). A secondary antibody, goat anti-mouse conjugated IR680 dye (Cat# A21057, Invitrogen; RRID:AB_141436) was diluted 1:5000 in a 1:1 mixture of PBS and Odyssey blocking buffer. Western blots were scanned on an Odyssey near-IR scanner (LI-COR Biosciences), and images were processed using Photoshop CE.

Ruehle S., Wager-Miller J., Straiker A., Farnsworth J., Murphy M.N., Loch S., Monory K., Mackie K, & Lutz B. (2017). Discovery and characterization of two novel CB1 receptor splice variants with modified N-termini in mouse. Journal of neurochemistry, 142(4), 521-533.

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Western Blot Analysis of Tight Junction Proteins

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Isolated epithelial cells were washed with DPBS containing sodium orthovanadate and lysed in 50 mmol/L HEPES, 250 mmol/L NaCl, 20 mmol/L β-glycerophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L EDTA, 1% NP-40, and protease inhibitors (Calbiochem). Lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis through 14% Tris-glycine gels (Invitrogen), transferred to polyvinylidene difluoride membranes, blocked (Odyssey Blocking Buffer, cat. 927-70001; Li-COR Biosciences), and incubated with anti–claudin-1 (1:150), anti–claudin-2 (1:1000), or anti–glyceraldehyde-3-phosphate dehydrogenase (1:2500, ab9485; Abcam) overnight at 4ºC followed by incubation with IRDye goat anti-rabbit antibody (1:5000, cat. 926-32211; Li-COR Biosciences). Images were visualized with the Odyssey Infrared Imaging System (Li-COR Biosciences). Expression levels for each protein were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD) and normalized to that of glyceraldehyde-3-phosphate dehydrogenase.

Shimodaira Y., More S.K., Hamade H., Blackwood A.Y., Abraham J.P., Thomas L.S., Miller J.H., Stamps D.T., Castanon S.L., Jacob N., Ha C.W., Devkota S., Shih D.Q., Targan S.R, & Michelsen K.S. (2023). DR3 Regulates Intestinal Epithelial Homeostasis and Regeneration After Intestinal Barrier Injury. Cellular and Molecular Gastroenterology and Hepatology, 16(1), 83-105.

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Immunofluorescence Staining of Cultured Cells

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Cells were cultured in the Lab-Tek II CC2 Chamber Slide System 4-well (Thermo Fisher Scientific, # 154917). After the indicated treatment, the cells were fixed and permeabilized in cold methanol for 10 min at −20 °C. Then, the slides were washed with 1 × PBS for 10 min and blocked with Odyssey Blocking Buffer (LI-COR Biosciences, # 927-40000) for 1 h. The slides were incubated in Odyssey Blocking Buffer with appropriately diluted primary antibodies at 4 °C for 16 h. After 3 washes (10 min per wash) with 1 × PBS, the cells were incubated with the corresponding Alexa Fluor conjugated secondary antibodies (Life Technologies) for 1 h at room temperature. The slides were washed three times (10 min each time) with 1 × PBS and counterstained with 300 nM DAPI for 1 min, followed by washing with 1 × PBS for 1 min. After air-drying, the slides were sealed with Gold Seal Cover Glass (Electron Microscopy Sciences, # 3223) using Fluoro-gel (Electron Microscopy Sciences, # 17985-10).

Wu Y., Song K., Hao W., Li J., Wang L, & Li S. (2022). Nuclear soluble cGAS senses double-stranded DNA virus infection. Communications Biology, 5, 433.

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Immunofluorescence Staining of Cultured Cells

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Cells were cultured in the Lab-Tek II CC2 Chamber Slide System 4-well (Thermo Fisher Scientific, # 154917). After the indicated treatment, the cells were fixed and permeabilized in cold methanol for 10 min at −20 °C. Then, the slides were washed with 1 × PBS for 10 min and blocked with Odyssey Blocking Buffer (LI-COR Biosciences, # 927-40000) for 1 h. The slides were incubated in Odyssey Blocking Buffer with appropriately diluted primary antibodies at 4 °C for 16 h. After 3 washes (5 min per wash) with 1 × PBS, the cells were incubated with the corresponding Alexa Fluor conjugated secondary antibodies (Life Technologies) for 1 h at room temperature. The slides were washed three times (5 min each time) with 1 × PBS and counterstained with 300 nM DAPI for 1 min, followed by washing with 1 × PBS for 1 min. After air-drying, the slides were sealed with Gold Seal Cover Glass (Electron Microscopy Sciences, # 3223) using Fluorogel (Electron Microscopy Sciences, # 17985-10). Images were captured and analysed using an Olympus Fluoview Confocal microscope (Olympus, Center Valley, PA).

Wang L., Fu B., Li W., Patil G., Liu L., Dorf M.E, & Li S. (2017). Comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction. Nature Communications, 8, 13876.

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