Startingblock buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

StartingBlock buffer is a protein-based blocking agent used to reduce non-specific binding in immunoassays and other protein-based applications. It is designed to effectively block unoccupied binding sites on membranes or solid supports, preventing the adsorption of non-target proteins and improving the specificity of the assay.

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Lab products found in correlation

Odyssey infrared imaging system, by LI COR (2 mentions) Superblock buffer, by Thermo Fisher Scientific (2 mentions) 1 step ultra tmb elisa, by Thermo Fisher Scientific (2 mentions) Infinite m200 plate reader, by Tecan (2 mentions) Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions) Rabbit anti gsk 3β, by Cell Signaling Technology (1 mentions) P gsk3β, by Cell Signaling Technology (1 mentions) β actin, by Abcam (1 mentions) Protein assay kit, by Bio-Rad (1 mentions) Phosphatase inhibitor cocktail, by Roche (1 mentions) Rabbit anti p65 antibody, by Cell Signaling Technology (1 mentions) Rabbit anti smad1 antibody, by Cell Signaling Technology (1 mentions) Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Iblot 2 dry blotting system, by Thermo Fisher Scientific (1 mentions) Laemmli sample buffer, by Bio-Rad (1 mentions) Ve cadherin, by Santa Cruz Biotechnology (1 mentions) Biorad western blot imaging system, by Bio-Rad (1 mentions) Pecam 1, by Abcam (1 mentions) Vegfr3, by R&D Systems (1 mentions) Vegfr2, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

StartingBlock T20 (PBS) Blocking Buffer StartingBlock Block Buffer StartingBlock T20 blocking buffer StartingBlock blocking buffer StartingBlock T20 (TBS) Blocking Buffer StartingBlock (TBS) Blocking Buffer StartingBlock PBS blocking buffer StartingBlock

15 protocols using startingblock buffer

Shear Stress-Induced NF-κB and Smad1 Activation

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Cells were seeded on tissue culture plastic slides cut from 150 mm tissue culture dishes (Falcon), coated with 20 μg.ml⁻¹ fibronectin. Confluent cells were subjected to steady laminar shear stress in a modified parallel plate flow chamber (Figure 1) in which the gasket was a silicon sheet of either 0.8 or 1.6 mm height (Grace Bio-Labs, Bend, OR, #664172 and #664283) cut to generate a linear gradient of shear stress, calculated from (Usami et al., 1993 (link)). Flow was applied for 16 hr in starvation medium. Cells were then fixed with 4% formaldehyde in PBS for 10 min, permeabilized with 0.5% Triton x-100 in PBS for 10 min, blocked with Startingblock buffer (ThermoScientific) for 30 min at room temperature and probed overnight at 4°C with a primary antibody diluted in Startingblock buffer. Slides were stained with Hoechst 33342 to label nuclei, with rabbit anti-p65 antibody (Cell Signaling) to label NF-κB, and with rabbit anti-Smad1 antibody (Cell Signaling).

Baeyens N., Nicoli S., Coon B.G., Ross T.D., Van den Dries K., Han J., Lauridsen H.M., Mejean C.O., Eichmann A., Thomas J.L., Humphrey J.D, & Schwartz M.A. (2015). Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point. eLife, 4, e04645.

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Propofol-Induced Neuronal Protein Signaling

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Following 6 h of propofol exposure, neurons were collected and lysed in N-PER Neuronal Protein Extraction Reagent (Pierce, Thermo Scientific) containing protease inhibitor and phosphatase inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Lysates were centrifuged at 10,000 g for 15 min at 4°C. The supernatant was collected and the concentration of each sample was determined using Bio-Rad Protein Assay Kit. The samples were denatured by boiling for 5 min at 97°C. A protein quantity of 15 μg/lane was loaded and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the gel were then transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was pre-blocked with Starting Block Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and then incubated with primary antibodies against rabbit phosphorylated Akt (p-Akt) (Ser-473), rabbit anti-Akt, rabbit anti-phosphorylated GSK3β (p-GSK3β) (Ser9), rabbit anti-GSK3β (Cell Signaling, Danvers, MA, USA), and β-actin (Abcam, Cambridge, MA, USA). After washing with Tris-buffered saline with Tween-20 (TBS-T), the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (cell signaling) for 1 h at room temperature. The densities of immunoblots were quantified using ImageJ software and the data were expressed as % control.

Liu Y., Yan Y., Inagaki Y., Logan S., Bosnjak Z.J, & Bai X. (2017). Insufficient Astrocyte-Derived BDNF Contributes to Propofol-Induced Neuron Death through Akt/GSK3β/Mitochondrial Fission Pathway. Anesthesia and analgesia, 125(1), 241-254.

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Western Blot Protein Analysis Protocol

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Protein samples (25–50 μg) were boiled in Laemmli sample buffer (BioRad, 161-0737) at 98 °C for 5 min, resolved on Bolt 4–12% Bis-Tris gels (Thermo Fisher Scientific, NW04120BOX) followed by transfer to nitrocellulose membrane using iBlot 2 dry blotting system (Thermo Fisher Scientific, IB21001). Membranes were incubated with StartingBlock buffer (Thermo Fisher, 37543) for 30 min followed by overnight incubation in primary antibodies at 4 °C. Membranes were washed with 1×Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and incubated with fluorophore-conjugated secondary antibodies (AlexaFluor-680 or AlexaFluor-800) for 1 h at room temperature. Then, membranes were washed three times with TBS-T, and images were captured using an Odyssey Infrared Imaging System (LI-COR Biosciences). For Drosophila, 10-day-old fly head lysates were isolated and prepared in Laemmli sample buffer for western blot analyses as previously described (16 (link)).

Abreha M.H., Ojelade S., Dammer E.B., McEachin Z.T., Duong D.M., Gearing M., Bassell G.J., Lah J.J., Levey A.I., Shulman J.M, & Seyfried N.T. (2021). TBK1 interacts with tau and enhances neurodegeneration in tauopathy. The Journal of Biological Chemistry, 296, 100760.

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Quantifying Tau Phosphorylation Levels

Cited 2 times

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Levels of total and phosphorylated tau were assessed using the Low-tau ELISA protocol previously published [1 (link), 22 (link)]. 96-well plates were coated for 48 h at 4 °C with specific purified monoclonal tau antibodies (DA31, CP13, PHF1, RZ3) at a concentration of 6 μg/ml. After washing, plates were blocked for 1 h at RT using StartingBlock buffer (Thermo Fisher Scientific, Waltham, MA). Brain samples and standards were diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBS and loaded on the plates. Once the samples were added, the total tau detection antibody DA9-HRP, diluted 1:50 in 20% SuperBlock in 1XTBS, was added to the samples and tapped to combine. Plates were then incubated overnight at 4 °C. Next day, 1-Step ULTRA TMB-ELISA (Thermo Fisher Scientific) was added for 30 min at RT, followed by 2 M H₂SO₄ to stop the reaction. Plates were read with Infinite m200 plate reader (Tecan, San Jose, CA) at 450 nm.

Vitale F., Giliberto L., Ruiz S., Steslow K., Marambaud P, & d’Abramo C. (2018). Anti-tau conformational scFv MC1 antibody efficiently reduces pathological tau species in adult JNPL3 mice. Acta Neuropathologica Communications, 6, 82.

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Immunoblotting analysis of angiogenic proteins

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Cells were washed with cold PBS and proteins extracted with Laemmli's buffer. Samples were run on 10 or 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with StartingBlock buffer (ThermoScientific) and probed with primary antibodies overnight at 4°C: VEGFR3 (R&D systems), phospho-VEGFR3 (Cell Applications), VEGFR2 (Cell Signaling), PECAM-1 (Abcam), VE-cadherin (Santa Cruz), GFP (Invitrogen) and actin (Santa Cruz). DyLight conjugated fluorescent secondary antibodies (680 nm and 800 nm, Thermoscientific) or HRP-conjugated antibodies were used to detect primary antibodies. Bands were detected and quantified with an Odyssey infrared imaging system for DyLight antibodies (Li-Cor) or a BioRad western blot imaging system (Bio Rad).

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Evaluating Ebola Glycoprotein Binding

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Filovirus glycoproteins lacking the transmembrane domain (GPΔTM), EBOV GP_CL, or EBOV sGP were coated onto 96-well Maxisorb ELISA plates at 100 ng/well diluted in DPBS overnight at 4°C. On the following day, the plates were washed three times with 300 μL of 1X DPBST (0.05% Tween-20) and blocked with StartingBlock Buffer (Thermo Scientific) for 1 hour at room temperature. After blocking, plates were washed as described above before adding CA45 diluted into 1X DPBST (0.05% Tween-20) at pH 7.5, 5.5 or 4.5 for 1 hour at room temperature. After incubation, plates were washed as described above and a 1:3000 dilution of goat anti-human-HRP (KPL) diluted in StartingBlock Buffer was added for 1 hour at room temperature before a final wash and the addition of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (Life Technologies) for 30 minutes. Plates were read at an optical density of 650 nm on a VersaMax plate reader. Softmax was used to fit the data to a 4PL curve.

Zhao X., Howell K.A., He S., Brannan J.M., Wec A.Z., Davidson E., Turner H.L., Chiang C.I., Lei L., Fels J.M., Vu H., Shulenin S., Turonis A.N., Kuehne A.I., Liu G., Ta M., Wang Y., Sundling C., Xiao Y., Spence J.S., Doranz B.J., Holtsberg F.W., Ward A.B., Chandran K., Dye J.M., Qiu X., Li Y, & Aman M.J. (2017). Immunization-elicited Broadly Protective Antibody Reveals Ebolavirus Fusion Loop as a Site of Vulnerability. Cell, 169(5), 891-904.e15.

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Quantifying Tau Protein Levels via ELISA

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High binding 96-well ELISA plates were coated with AT8 (2 μg/ml) (Thermo Fisher Scientific, MN1020) in PBS overnight. The plates were washed with TBST and blocked with StartingBlock buffer (Thermo Fisher Scientific, 37538) for 1 h at RT. The medium and cell lysate were diluted with SuperBlock (Thermo Fisher Scientific, 37516) and loaded on plates for 2 h incubation followed by 1 h incubation with Biotin-HT7 (Thermo Fisher Scientific MN1000B, 1:200). The AT8-positive signals were detected by streptavidin-HRP followed by chromogenic reaction with TMB substrate and reading by Biotek Synergy HT microplate reader at 450 nm.

Wang C., Fan L., Khawaja R.R., Liu B., Zhan L., Kodama L., Chin M., Li Y., Le D., Zhou Y., Condello C., Grinberg L.T., Seeley W.W., Miller B.L., Mok S.A., Gestwicki J.E., Cuervo A.M., Luo W, & Gan L. (2022). Microglial NF-κB drives tau spreading and toxicity in a mouse model of tauopathy. Nature Communications, 13, 1969.

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Western Blot Protein Detection

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Purified proteins or protein extracts were separated under denaturing conditions by SDS–PAGE and transferred to nitrocellulose membranes using Trans-Blot Turbo nitrocellulose transfer pack (Bio-Rad, 1704159). Membranes were blocked for 45 min at room temperature using 1x StartingBlock buffer (Thermo Fisher Scientific, 37578). After blocking, membranes were incubated overnight with the appropriate primary antibody (1:1000 in PBS), washed three times in TBST, and probed for 40 min using a fluorophore-conjugated secondary antibody diluted 1:5000 in the blocking buffer. Membranes were washed three times using TBST, over 15 min, and scanned using an Odyssey infrared imaging system (LI-COR Biosciences).

Miihkinen M., Grönloh M.L., Popović A., Vihinen H., Jokitalo E., Goult B.T., Ivaska J, & Jacquemet G. (2021). Myosin-X and talin modulate integrin activity at filopodia tips. Cell Reports, 36(11), 109716.

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Quantifying Tau Protein Levels in Brain Samples

Cited 2 times

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Levels of total and phosphorylated tau were assessed using the Low-tau ELISA (enzyme-linked immunosorbent assay) protocol previously published [64 (link), 65 (link)]. 96-well plates were coated for 48 h at 4 °C with specific purified monoclonal tau antibodies (DA31, CP13, PHF1, RZ3) at a concentration of 6 μg/ml. After washing, plates were blocked for 1 h at RT using StartingBlock buffer (Thermo Fisher Scientific). Brain samples and standards were diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBS and loaded on the plates. Once the samples were added, the total tau detection antibody DA9-HRP, diluted 1:50 in 20% SuperBlock in 1XTBS, was added to the samples and tapped to combine. Plates were then incubated overnight at 4 °C. Next day, 1-Step ULTRA TMB-ELISA (Thermo Fisher Scientific) was added for 30 min at RT, followed by 2 N H₂SO₄ to stop the reaction. Plates were read with Infinite m200 plate reader (Tecan, San Jose, CA) at 450 nm.

Vitale F., Ortolan J., Volpe B.T., Marambaud P., Giliberto L, & d’Abramo C. (2020). Intramuscular injection of vectorized-scFvMC1 reduces pathological tau in two different tau transgenic models. Acta Neuropathologica Communications, 8, 126.

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LIPA Protein Expression Analysis in NSCs

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NSCs were seeded on 10-cm dishes with 10 μM ROCK inhibitor-supplemented media. The next day media was changed to remove ROCK inhibitor. Media was changed again the following day, and the cells were grown for another 48 h. Cells were harvested using M-PER™ Mammalian Protein Extraction Reagent (ThermoFisher) containing protease inhibitors (Roche) with scraping. Cell debris was pelleted at 13,200 × g for 5 min at 4 °C, and protein content was quantitated in the lysates. 15 μg of lysate from each cell line was resolved on a 4–12% NuPAGE Novex gel (ThermoFisher), and transferred using the iBlot Dry Blotting System (ThermoFisher). Blots were blocked with Starting Block buffer (ThermoFisher), incubated with primary antibody mouse-anti-LIPA clone 9G7F12,7G6D7 (ThermoFisher), and with secondary antibody goat-anti-mouse-HRP (Cell Signaling Technology, CST), then developed. Blots were then stripped and GAPDH expression was evaluated as a loading control with rabbit-anti-GAPDH (CST) primary antibody and goat-anti-rabbit-HRP (CST) secondary antibody.

Aguisanda F., Yeh C.D., Chen C.Z., Li R., Beers J., Zou J., Thorne N, & Zheng W. (2017). Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics. Orphanet Journal of Rare Diseases, 12, 120.

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