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Neutravidin

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Neutravidin is a streptavidin-based reagent used in various bioanalytical techniques. It offers high-affinity binding to biotin, facilitating the detection and separation of biotinylated molecules. Neutravidin is a versatile tool for researchers across multiple disciplines.

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Lab products found in correlation

Neutravidin, by Thermo Fisher Scientific (5 mentions) Nap 5 size exclusion column, by GE Healthcare (4 mentions) Alexa fluor 647 nhs ester, by Thermo Fisher Scientific (4 mentions) Nanodrop 2000c uv vis spectrophotometer, by Thermo Fisher Scientific (4 mentions) Hepes buffer, by Cytiva (1 mentions) Prism 5, by GraphPad (1 mentions) Cm5 sensor chip, by GE Healthcare (1 mentions) Biacore s200 instrument, by GE Healthcare (1 mentions)

7 protocols using neutravidin

1

Neutravidin-AlexaFluor647 Conjugate Preparation

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A reaction mixture was assembled in a 1.5 mL Eppendorf tube with following components (added in this order): 200 μL of 5 mg/mL Neutravidin (Life Technologies) in PBS, 20 μL of 1 M sodium bicarbonate in water, and 10 μL of 10 mg/mL AlexaFluor647-NHS Ester (Life Technologies) in anhydrous DMSO. The tube was incubated at room temperature with rotation in the dark for 3 h. The Neutravidin-AlexaFluor647 conjugate was purified from unreacted dye using a NAP-5 size-exclusion column (GE Healthcare Life Sciences) according to the manufacturer’s instructions. The conjugate was typically eluted from the column in 500 μL cold PBS. Absorbance values, determined using a Nanodrop 2000c UV-vis spectrophotometer (Thermo Scientific), were typically as follows: A280 = ~0.284 and A647 = ~1.625. The conjugate was stable at 4 °C in the dark for at least 4 months and was flash frozen and stored at −80 °C for longer term. For mammalian cell labeling experiments, the conjugate was diluted 1000-fold in PBS containing 1% BSA.
Martell J.D., Yamagata M., Deerinck T.J., Phan S., Kwa C.G., Ellisman M.H., Sanes J.R, & Ting A.Y. (2016). A split horseradish peroxidase for detection of intercellular protein-protein interactions and sensitive visualization of synapses. Nature biotechnology, 34(7), 774-780.
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2

SPR Biacore Protocol for Aptamer Binding

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SPR Biacore studies were carried out on a CM7 (GE Healthcare) chip with neutravidin custom immobilized on the surface. The surface was activated with a mixture of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (0.2 M) and N-hydroxysuccinimide (0.05 M) for 420 sec at 10 μL/min. neutravidin (10 mg/mL; Thermo Scientific) was dissolved in HyClone water then diluted 1/10 in 10 mM sodium acetate buffer (pH = 4.5) and added at 30 μL/min for 210 sec. The surface was blocked with 1 M ethanolamine for 600 sec at 5 μL/min. 3′-biotin-T10 4A018 aptamer was heated to 95°C then introduced at 5 μM in 5 mM MgSO4 for 180 sec at 30 μL/min. Samples were diluted to 27.3 μM in HEPES buffer from Biacore (10 mM HEPES, 150 mM NaCl, 0.05% Surfactant P20) and dialyzed into HEPES buffer overnight. The samples were serially diluted to appropriate concentrations then introduced onto the chip at 30 μL/min for 30 sec. Data analysis was performed with BIAevaluation software for three experiments of three replicates for each compound with the background subtracted data (reference channel 1) and plotted in GraphPad Prism 5 using a one site total binding model. Each experiment included freshly prepared reagents introduced onto the same chip in triplicate.
Martin J.A., Mirau P.A., Chushak Y., Chávez J.L., Naik R.R., Hagen J.A, & Kelley-Loughnane N. (2015). Single-Round Patterned DNA Library Microarray Aptamer Lead Identification. Journal of Analytical Methods in Chemistry, 2015, 137489.
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3

Neutravidin-AlexaFluor647 Conjugation

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A reaction mixture was assembled in a 1.5 mL Eppendorf tube with the following components (added in this order): 200 μL of 5 mg/mL Neutravidin (Life Technologies) in PBS, 20 μL of 1 M sodium bicarbonate in water, and 10 μL of 10 mg/mL AlexaFluor647-NHS Ester (Life Technologies) in anhydrous DMSO. The tube was incubated at room temperature with rotation in the dark for 3 h. The Neutravidin-AlexaFluor647 conjugate was purified from unreacted dye using a NAP-5 size-exclusion column (GE Healthcare Life Sciences) according to the manufacturer’s instructions. The conjugate was typically eluted from the column in 500 μL cold PBS. Absorbance values, determined using a Nanodrop 2000c UV-vis spectrophotometer (Thermo Scientific), were typically as follows: A280 = ~0.284 and A647 = ~1.625. The conjugate was stable at 4 °C in the dark for at least 4 months and was flash frozen and stored at −80 °C for longer term storage. For mammalian cell labeling experiments, the conjugate was diluted 1,000-fold in PBS containing 1% BSA.
Branon T.C., Bosch J.A., Sanchez A.D., Udeshi N.D., Svinkina T., Carr S.A., Feldman J.L., Perrimon N, & Ting A.Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature biotechnology, 36(9), 880-887.
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4

Efficient Substrate Annealing and Purification

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All oligonucleotides were purchased from Integrated DNA Technologies (IDT) and were HPLC-purified by the manufacturer. All oligonucleotides used for generating RNA-containing substrates were purified by the manufacturer via RNase-free HPLC purification.
Substrates were annealed by mixing their component oligonucleotides at specific ratios in a buffer containing 50 mM TRIS pH 8.0, 10 mM EDTA, and 100 mM NaCl (TE 100) and then heated at 95 °C for 5 min, followed by slow cooling to room temperature in a thermocycler. The oligonucleotide mixing ratios used for substrate annealing before gel purification are indicated in Supplementary Fig. 7 for each substrate. Throughout the manuscript, substrates are numbered according to the codes presented in Supplementary Fig. 7. All substrates were purified on 10% non-denaturing TBE-PAGE gels and recovered from gel by the crush and soak method in TE 100 for 30 min at 16 °C and vigorous shaking. Substrates used in the experiments presented in the current manuscript exhibited >80% purity. For the bulk experiments that used blocked biotinylated substrates, a 2.5× molar excess of tetrameric NeutrAvidin (GE Healthcare) was added to the substrates and incubated for 5 min prior to the experiments.
Raducanu V.S., Tehseen M., Al-Amodi A., Joudeh L.I., De Biasio A, & Hamdan S.M. (2022). Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics. Nature Communications, 13, 6973.
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5

Neutravidin-AlexaFluor647 Conjugate Preparation

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A reaction mixture was assembled in a 1.5 mL Eppendorf tube with following components (added in this order): 200 μL of 5 mg/mL Neutravidin (Life Technologies) in PBS, 20 μL of 1 M sodium bicarbonate in water, and 10 μL of 10 mg/mL AlexaFluor647-NHS Ester (Life Technologies) in anhydrous DMSO. The tube was incubated at room temperature with rotation in the dark for 3 h. The Neutravidin-AlexaFluor647 conjugate was purified from unreacted dye using a NAP-5 size-exclusion column (GE Healthcare Life Sciences) according to the manufacturer’s instructions. The conjugate was typically eluted from the column in 500 μL cold PBS. Absorbance values, determined using a Nanodrop 2000c UV-vis spectrophotometer (Thermo Scientific), were typically as follows: A280 = ~0.284 and A647 = ~1.625. The conjugate was stable at 4 °C in the dark for at least 4 months and was flash frozen and stored at −80 °C for longer term. For mammalian cell labeling experiments, the conjugate was diluted 1000-fold in PBS containing 1% BSA.
Martell J.D., Yamagata M., Deerinck T.J., Phan S., Kwa C.G., Ellisman M.H., Sanes J.R, & Ting A.Y. (2016). A split horseradish peroxidase for detection of intercellular protein-protein interactions and sensitive visualization of synapses. Nature biotechnology, 34(7), 774-780.
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6

Biacore Assay for KRAS/CRAF Binding

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SPR binding experiments were performed on a Biacore S200 instrument (GE). Avi-KRASG12D (amino acids 2–188) loaded with the non-hydrolyzable GTP analog, GppNHp, or avi-tagged CRAF-RBD (amino acids 52–131) were captured on CM5 sensor chips (GE) containing amine coupled Neutravidin (10000 RU). For single does binding, compounds were diluted to 100 μM in buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.05% Tween 20, 5 mM MgCl2, and 2.5% DMSO prior to injection over avi-CRAF-RBD (3,000 RU) or over avi-KRASG12D-GppNHp (3,800 RU). For dose response binding, a 2-fold dilution series of each compound was prepared (100–1.56 μM in the above buffer) and injected over avi-RAF-RBD (1,785 RU) or over avi-KRASG12D-GppNHp (2,300 RU). The data were processed by subtracting binding responses on the reference flow cells as well as binding responses when buffer was injected. The samples were also corrected for DMSO mismatches using a DMSO standard curve.
Durrant D.E., Smith E.A., Goncharova E.I., Sharma N., Alexander P.A., Stephen A.G., Henrich C.J, & Morrison D.K. (2021). Development of a High-throughput NanoBRET Screening Platform to Identify Modulators of the RAS/RAF Interaction. Molecular Cancer Therapeutics, 20(9), 1743-1754.
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7

Neutravidin-AlexaFluor647 Conjugation

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A reaction mixture was assembled in a 1.5 mL Eppendorf tube with the following components (added in this order): 200 μL of 5 mg/mL Neutravidin (Life Technologies) in PBS, 20 μL of 1 M sodium bicarbonate in water, and 10 μL of 10 mg/mL AlexaFluor647-NHS Ester (Life Technologies) in anhydrous DMSO. The tube was incubated at room temperature with rotation in the dark for 3 h. The Neutravidin-AlexaFluor647 conjugate was purified from unreacted dye using a NAP-5 size-exclusion column (GE Healthcare Life Sciences) according to the manufacturer’s instructions. The conjugate was typically eluted from the column in 500 μL cold PBS. Absorbance values, determined using a Nanodrop 2000c UV-vis spectrophotometer (Thermo Scientific), were typically as follows: A280 = ~0.284 and A647 = ~1.625. The conjugate was stable at 4 °C in the dark for at least 4 months and was flash frozen and stored at −80 °C for longer term storage. For mammalian cell labeling experiments, the conjugate was diluted 1,000-fold in PBS containing 1% BSA.
Branon T.C., Bosch J.A., Sanchez A.D., Udeshi N.D., Svinkina T., Carr S.A., Feldman J.L., Perrimon N, & Ting A.Y. (2018). Efficient proximity labeling in living cells and organisms with TurboID. Nature biotechnology, 36(9), 880-887.
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