Neutravidin slurry

Manufactured by Thermo Fisher Scientific

Sourced in United States

NeutrAvidin slurry is a laboratory product used for affinity-based separation techniques. It contains avidin-coated agarose beads suspended in a buffer solution. NeutrAvidin, a deglycosylated form of avidin, binds strongly and specifically to biotin, enabling the capture and purification of biotinylated molecules from complex samples.

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Lab products found in correlation

Ez link sulfo nhs ss biotin solution, by Thermo Fisher Scientific (1 mentions) Ez link sulfo biotin, by Thermo Fisher Scientific (1 mentions) Nelfe, by Abcam (1 mentions) Vinculin, by Merck Group (1 mentions) Total rhoa, by Santa Cruz Biotechnology (1 mentions) Fibronectin fn, by BD (1 mentions) Grp78, by BD (1 mentions)

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NeutrAvidin (263 citations)

5 protocols using neutravidin slurry

Cell Surface Biotinylation of Neuronal Cultures

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Mixed cortical-hippocampal neuronal cultures prepared from newborn WT or S2KO mice were plated on poly-D-lysine-coated plates and cell surface biotinylation was performed at DIV10-12. To do so, cells were cooled down on ice for 15 min and washed 3 times with PBS-Mg-Ca (PBS supplemented with 0.5 mM MgCl₂ and 1 mM CaCl₂). Next, cells were incubated on ice with EZ-Link Sulfo-NHS-SS-Biotin solution (Thermo Fisher Scientific; 0.5 mg/ml in PBS) for 25 min, washed 3 times with quenching buffer (40 mM glycine, 0.4% BSA in TBS-Ca-Mg) and 2 times with TBS-Ca-Mg. Cells were scraped in lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 2 mM EGTA, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, protease and phosphatase inhibitors) and the lysates were rotated for 1.5 hours at 4°C. After centrifugation (15 min, 14,000x g), the supernatants were used for pull down of biotinylated proteins with NeutrAvidin slurry (Thermo Fisher Scientific). Equal amounts of proteins were loaded on the slurry. After washing 3 times with lysis buffer, the beads were snap-frozen and stored at −80°C until mass spectrometry analysis or boiled in Laemmli buffer to release captured proteins for western blot analysis.

Malik A.R., Szydlowska K., Nizinska K., Asaro A., van Vliet E.A., Popp O., Dittmar G., Fritsche-Guenther R., Kirwan J.A., Nykjaer A., Lukasiuk K., Aronica E, & Willnow T.E. (2019). SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology. Cell Reports, 26(10), 2792-2804.e6.

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Biotin-labeling and Affinity Purification

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After treatment, cells were incubated with 1 mg/mL EZ-link Sulfo-Biotin (Thermo Fisher, Waltham, MA, USA) for 30 min, then washed with 0.1 M glycine in PBS to remove excess Sulfo-Biotin, lysed, and clarified, and equal quantities of protein were incubated overnight in a 50% Neutravidin slurry (ThermoFisher, Waltham, MA, USA) to capture biotin-tagged proteins. Beads were washed 5× with lysis buffer and bound proteins cleaved by boiling for 10 min in 2× PSB. Samples were separated using SDS-PAGE and immunoblotted.

Trink J., Li R., Palarasah Y., Troyanov S., Andersen T.E., Sidelmann J.J., Inman M.D., Pizzo S.V., Gao B, & Krepinsky J.C. (2021). Activated Alpha 2-Macroglobulin Is a Novel Mediator of Mesangial Cell Profibrotic Signaling in Diabetic Kidney Disease. Biomedicines, 9(9), 1112.

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mRNA-Protein Interactome Profiling

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Hep3B or Huh1 HCC cells were cultured on 15 cm plates until 80% confluent. Cells were then washed 2× with ice cold 1× PBS on ice. 1mL of RIPA buffer supplemented with 1mM of PMSF was then added onto plates and incubated on ice for 10 min. Cells were then scraped and collected followed by a 10 min incubation on ice. Lysates were centrifuge for 10 min at 13K RPM for 10 min at 4C. The supernatant was then transferred into a new microcentrifuge tube. For IP, 50 μl of neutravidin slurry (Thermo Scientific) were washed twice with 1× PBS. The slurry was then centrifuged for 1 min at 1500 RPM and the supernatant was removed via pipetting. 8 μl of 100 μM mRNA oligos (HPLC grade generated by IDT) were then incubated with the slurry and 100ul of 1× TBS for 2 hr, washed 3× with 1×TBS and incubated with 150ul of whole lysates freshly prepared and incubated overnight in 4 °C with rotation. 4 μl (~2.5%) of the lysates were used as input. The slurry was then washed and immuno-blotting was performed to test for binding (NELFE from Abcam (cat. ab170104)). For oligo sequence, see Supplemental Materials.

Dang H., Takai A., Forgues M., Pomyen Y., Mou H., Xue W., Ray D., Ha K.C., Morris Q.D., Hughes T.R, & Wang X.W. (2017). Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma. Cancer cell, 32(1), 101-114.e8.

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Biotin-Based Protein Enrichment

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After treatment, MCs were incubated with 1 mg/mL EZ-linked Sulfo-Biotin (Pierce, Waltham, MA, USA) for 30 min. Excess Sulfo-Biotin was removed by washes with 0.1 M glycine in PBS, then cells were lysed, clarified, and normalized concentrations of proteins were incubated overnight with a 50% Neutravidin slurry (Fisher) to capture biotin-tagged proteins. The following day, beads were washed with lysis buffer 5 times, after which tagged proteins were cleaved from beads by boiling for 10 min in 2× PSB. Samples were separated using SDS-PAGE prior to immunoblotting.

Trink J., Li R., Squire E., O’Neil K., Zheng P., Gao B, & Krepinsky J.C. (2022). Integrin β1/Cell Surface GRP78 Complex Regulates TGFβ1 and Its Profibrotic Effects in Response to High Glucose. Biomedicines, 10(9), 2247.

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Biotin-mediated Protein Pulldown Assay

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Whole-cell lysates were collected as previously described [8 (link)]. For biotinylation experiments, MC were incubated with 1 mg/mL EZ-linked Sulfo-Biotin (Pierce) for 30 min followed by 0.1 M glycine in PBS to remove excess Sulfo-Biotin, then whole-cell lysates were collected and incubated overnight in a 50% NeutrAvidin slurry (Fisher) to pull down biotin-tagged proteins. Immunoprecipitates were assessed with SDS–PAGE and immunoblotted for surface proteins. The following antibodies were used for immunoblotting: α-smooth muscle actin (αSMA) (Pierce, 1:5000), phosphorylated myosin phosphatase target (pMYPT) Thr696 (Cell Signaling; 1:1000), fibronectin (FN) (BD Biosciences, 1:1000), total RhoA (Santa Cruz, 1:500), MTJ-1 (Santa Cruz, 1:1000), GRP78 (BD Biosciences, 1:1000), PDGFRβ (Cedarlane, 1:1000), vinculin (Sigma, 1:1000) and tubulin (Santa Cruz, 1:5000).

Soomro A., Trink J., O’Neil K., Li R., Naiel S., Gao B., Ask K, & Krepinsky J.C. (2021). Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease. International Journal of Molecular Sciences, 22(6), 2839.

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