Intercept protein free blocking buffer

Manufactured by LI COR

Sourced in United States

Intercept Protein-Free Blocking Buffer is a high-performance buffer designed for blocking non-specific binding in western blotting and other immunoassays. It is a ready-to-use solution that effectively blocks membranes without adding protein or other interfering substances.

Automatically generated - may contain errors

Lab products found in correlation

Odyssey fc imaging system, by LI COR (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Irdye 800cw donkey anti mouse, by LI COR (1 mentions) Laemelli buffer, by Bio-Rad (1 mentions) Chameleon duo pre stained protein ladder, by LI COR (1 mentions) Odyssey machine, by LI COR (1 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions) Mini trans blot apparatus, by Bio-Rad (1 mentions) M csf, by R&D Systems (1 mentions) Protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Empiria studio software, by LI COR (1 mentions) Pierce bicinchoninic acid assay kit, by Thermo Fisher Scientific (1 mentions) Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions) Immobilon fl pvdf membrane, by Merck Group (1 mentions) Loading buffer, by LI COR (1 mentions) Cellytic, by Merck Group (1 mentions) Criterion sds page gel, by Bio-Rad (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Image studio software, by LI COR (1 mentions) Irdye secondary antibody, by LI COR (1 mentions)

7 protocols using intercept protein free blocking buffer

Western Blot Analysis of Bacterial Proteins

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Bacteria for Western blots were grown overnight in TSB with or without dextrose. The following morning 1 mL of culture was centrifuged at 13,000 × g for 1 min, the supernatant was removed, and was heat inactivated at 70°C for 10 min. The supernatants were diluted 1:4 with Laemelli buffer (Bio-Rad) with 2-mercaptoethanol (Fischer Scientific) and boiled at 99 C for 10 min. 40 μL of boiled supernatant was loaded onto a Mini Protean TGX 12% gel (Bio-Rad) and run on a Mini Trans-Blot apparatus (Bio-Rad) for 90 min at 150 V with a Chameleon Duo Pre-Stained Protein ladder (LI-COR). The gel was transferred to a supported nitrocellulose 0.22 μm membrane (Bio-Rad) for 60 min at 100 V. The membranes were blocked for 4 h with Intercept protein-free blocking buffer (LI-COR), and then incubated overnight with the Abcam mouse MAb anti-alphahemolysim antibody (ab190467) at 1 μg/mL. The following day, the membranes were washed for 10 min, then 5 min, then 5 min with PBS 0.05% Tween (Sigma-Aldrich). The membranes were then stained with the secondary antibody IR Dye 800CW Donkey anti-mouse (LI-COR) diluted in donkey serum (Sigma-Aldrich) for 1 h, then washed as above. The membranes were imaged on a LI-COR Odyssey machine with 7 (800) and 2 (700) gains on the fluorescent channels.

Stephens A.C., Thurlow L.R, & Richardson A.R. (2022). Mechanisms Behind the Indirect Impact of Metabolic Regulators on Virulence Factor Production in Staphylococcus aureus. Microbiology Spectrum, 10(4), e02063-22.

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Assessing Protein Expression via Western Blot

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For the assessment of protein expression through western blot analysis, cells were cultured in 100‐mm tissue culture dishes with appropriate media type and allowed to reach ≈75 % confluency. Cells were then dosed with IC₂₀ concentrations of Ru-IM (1). Following 6h and 24h incubations with serum-free media, cell lysates were collected with the use of RIPA buffer and protein concentration quantification was completed with a Bradford assay. Western blot was carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred onto a polyvinylidene difluoride (PDVF) membrane (150 mA, 1h). Membranes were dried completely and then blocked with Intercept Protein-Free Blocking Buffer (LICOR Biosciences, Lincoln, NE, USA) for 1h on a shaking platform. Membranes were then incubated overnight on a shaking platform at 4°C 1:200 with primary antibody P53-DO1 (Santa Clara Biotechnology, Dallas, TX, USA) or 1:1000 with M-CSF (R&D Systems), and with control primary antibody total H4 or alpha tubulin. Membranes were then washed five times with TBST and incubated with anti-rabbit and anti-mouse antibodies (LICOR Biosciences) for 1h at room temperature. Following five TBST and one TBS wash, signals were visualized with 600 and 700 channels with an Odyssey FC imaging system (LICOR Biosciences).

Nayeem N., Yeasmin A., Cobos S.N., Younes A., Hubbard K, & Contel M. (2021). Investigation of the effects and mechanisms of anticancer action of a Ru(II)-arene iminophosphorane compound in triple negative breast cancer cells. ChemMedChem, 16(21), 3280-3292.

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Western Blot Analysis of ARID1B Protein

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Protein was extracted using Pierce RIPA Buffer (ThermoFisher, Waltham, MA) with Protease & Phosphatase Inhibitor Cocktail (ThermoFisher, Waltham, MA). Protein concentrations were measured using Pierce Bicinchoninic acid assay kit (ThermoFisher, Waltham, MA). Forty micrograms of protein lysate was separated on a 4–20% Stacking TGX Gels (BioRad, Hercules, CA) and transferred overnight at 30 V onto Immobilon-FL PVDF membrane (Millipore Sigma, Burlington, MA). PVDF membranes were blocked for 1 h with Intercept Protein-Free Blocking Buffer (LI-COR, Lincoln, NE). Following blocking, PVDF membranes were incubated with ms-Arid1b (1:1000, Abcam, ab47561) and rb-beta actin (1:2000, Millipore Sigma, SAB5600204) in Intercept for 2 h at room temperature. Following incubation, membranes were washed with Tris-buffered saline with Tween (TBST) 3× for 5 min. Membranes were then incubated with Goat anti-rabbit LI-COR 680 (1:2000) and goat anti-mouse LI-COR 800 (1:2000) in Intercept TBS for 1 h at RT. Following incubation, membranes were washed 3× with TBST before storing in 1× TBS. Membranes were imaged on Odyssesy CLx imager (LI-COR, Lincoln, NE). Quantitative analysis was performed using Empiria Studio software (LI-COR, Lincoln, NE).

Ellegood J., Petkova S.P., Kinman A., Qiu L.R., Adhikari A., Wade A.A., Fernandes D., Lindenmaier Z., Creighton A., Nutter L.M., Nord A.S., Silverman J.L, & Lerch J.P. (2021). Neuroanatomy and behavior in mice with a haploinsufficiency of AT-rich interactive domain 1B (ARID1B) throughout development. Molecular Autism, 12, 25.

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Western Blot Analysis of Protein Expression

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Cells were lysed using CelLytic (Sigma, St. Louis, MO) and tissue was lysed using RIPA buffer (EMD Millipore, Billerica, MA) and protease inhibitor cocktail (Fischer Scientific, Waltham, MA). Protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). Samples were diluted in water and loading buffer (LI-COR, Lincoln, NE), electrophoretically separated under denaturing conditions on 4–15% SDS-PAGE Criterion gels (Bio-Rad), and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in Intercept Protein-Free Blocking Buffer (LI-COR), followed by an overnight incubation with primary antibody for UCP1 (Invitrogen, Carlsbad, CA; rabbit polyclonal, #PA1-24894), ITA7 (Novus, Centennial CO; rabbit polyclonal, #NBP1-74207) or GAPDH (Santa Cruz; mouse monoclonal, #sc-32233) diluted in Intercept® Protein-Free Antibody Diluent (LI-COR). Membranes were incubated with appropriate IRDye secondary antibodies (LI-COR), and immunodetection was performed using near-infrared Odyssey Fc Imaging System (LI-COR). Images were obtained with Image Studio Software (LI-COR).

Gonzalez Porras M.A., Stojkova K., Vaicik M.K., Pelowe A., Goddi A., Carmona A., Long B., Qutub A.A., Gonzalez A., Cohen R.N, & Brey E.M. (2021). Integrins and extracellular matrix proteins modulate adipocyte thermogenic capacity. Scientific Reports, 11, 5442.

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Primary Mouse Astrocyte Protein Extraction

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Primary mouse astrocytes (passage 11) were isolated from mouse ONH as we previously described [90 (link)], and were grown to confluency in 6-well plates. Cells were rinsed with DPBS, and RIPA buffer (200 µl/well) supplemented with protease/phosphatase cocktail inhibitors (MilliporeSigma #11,836,170,001; #4,906,845,001) was added for 10 min on ice with gentle rocking. The cell extract was collected and centrifuged at 14000×g for 10 min and protein concentration measured by BCA assay. Samples were loaded onto a polyacrylamide gel (25 µg/well) and resolved with constant voltage at 200 V for 45 min. Resolved samples from the gel were transferred to PVDF and blocked in the Intercept protein-free blocking buffer (LI-COR, #927–65,001) for 1 h at room temperature. Primary antibody (see Table 1) was added in the incubation buffer (same blocking buffer with addition of 0.2% Tween) and incubated overnight at 4 degrees Celsius. Secondary antibody was applied for 1 hour in incubation buffer at room temperature. Fluorescent signal was detected using the Odyssey Licor System.

Mavlyutov T.A., Myrah J.J., Chauhan A.K., Liu Y, & McDowell C.M. (2022). Fibronectin extra domain A (FN-EDA) causes glaucomatous trabecular meshwork, retina, and optic nerve damage in mice. Cell & Bioscience, 12, 72.

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BambL-receptor Complexes Visualized

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BambL-receptor complexes were extracted, separated by SDS-PAGE (different acrylamide densities for different targets), and transferred onto nitrocellulose as before. After transfer, the membrane was blocked in Intercept Protein-Free Blocking Buffer (Li-Cor), and then incubated shaking in a 25 µg/mL (0.9 µM) solution of BambL-700 in the same buffer for 1 h at RT. The membrane was washed extensively and imaged with the Odyssey CLx in both channels. The membrane was then processed in immunoblots as before, using fluorescently labeled secondary antibodies and recording their bands in the 800-nm channel. In addition, a second recording of the 700-nm channel was taken. By comparing the pictures before and after antibody staining, we eliminated the possibility of false-positive hits which could have been introduced by BambL binding to glycans of the detection antibodies.

Frensch M., Jäger C., Müller P.F., Tadić A., Wilhelm I., Wehrum S., Diedrich B., Fischer B., Meléndez A.V., Dengjel J., Eibel H, & Römer W. (2021). Bacterial lectin BambL acts as a B cell superantigen. Cellular and Molecular Life Sciences, 78(24), 8165-8186.

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Western Blot Analysis of Protein Expression

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Brain and liver homogenates were generated as detailed above. BMDMs cultured in 96-well format were washed 2x with PBS and then collected in 25uL 1x CST Buffer (Cell Signaling, #9803S) supplemented with PMSF. 1mg/mL tissue/cell lysates were prepared for SDS-PAGE by diluting with 4X NuPage Sample Buffer (Invitrogen #NP007), 10X NuPage Reducing Buffer (Invitrogen #NP009), and RIPA Buffer (Teknova #R379). Samples were boiled for 5 min, and then run on a 4–12% Bis-Tris gel (Invitrogen #NP0322) in MES buffer (Invitrogen #NP0002), 120V for 1.5 hours. Gel was then transferred on the Bio-Rad semi-dry transfer apparatus mixed molecule weight program onto nitrocellulose. Transferred blot was blocked with Intercept protein-free blocking buffer (Licor, #927–80001). Blot was then probed for GCase (Sigma #G4171, 1:1000) and GAPDH (Sigma #G8795, 1:2500) for liver and brain tissues or Cathepsin D (Abcam #ab78552, 1:1000) and beta-Actin (Sigma #A2228, 1:3000) for BMDM cell lysates and detected via fluorescent secondaries (Licor) while incubating in LI-COR Intercept Blocking Buffer. After washing, blot was imaged and quantified on the Odyssey scanner, and analyzed in Prism by one-way ANOVA and Dunnett’s multiple comparison test.

Logan T., Simon M.J., Rana A., Cherf G.M., Srivastava A., Davis S.S., Yoon Low R.L., Chiu C.L., Fang M., Huang F., Bhalla A., Llapashtica C., Prorok R., Pizzo M.E., Calvert M.E., Sun E.W., Hsiao-Nakamoto J., Rajendra Y., Lexa K.W., Srivastava D.B., van Lengerich B., Wang J., Robles-Colmenares Y., Kim D.J., Duque J., Lenser M., Earr T.K., Nguyen H., Chau R., Tsogtbaatar B., Ravi R., Skuja L.L., Solanoy H., Rosen H.J., Boeve B.F., Boxer A.L., Heuer H.W., Dennis M.S., Kariolis M.S., Monroe K.M., Przybyla L., Sanchez P.E., Meisner R., Diaz D., Henne K.R., Watts R.J., Henry A.G., Gunasekaran K., Astarita G., Suh J.H., Lewcock J.W., DeVos S.L, & Di Paolo G. (2021). Rescue of a lysosomal storage disorder caused by Grn loss-of-function with a brain penetrant progranulin biologic. Cell, 184(18), 4651-4668.e25.

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