High capacity streptavidin agarose beads

Manufactured by Thermo Fisher Scientific

Sourced in United States

High-capacity streptavidin agarose beads are a type of affinity chromatography resin. They are composed of streptavidin, a protein with a high affinity for biotin, immobilized on agarose beads. These beads can be used to capture and purify biotinylated biomolecules from complex samples.

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Lab products found in correlation

Protein a g agarose bead, by Roche (2 mentions) Iodoacetamide, by GE Healthcare (1 mentions) Arg c, by Promega (1 mentions) 660 nm protein assay kit, by Thermo Fisher Scientific (1 mentions) Ms grade trypsin, by Promega (1 mentions) 13c615n4 arginine, by Thermo Fisher Scientific (1 mentions) 13c615n2 lysine, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase hrp conjugated streptavidin, by Thermo Fisher Scientific (1 mentions) Hrp conjugated species specific secondary antibodies, by Cell Signaling Technology (1 mentions) Rpmi 1640 medium for silac, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Supersignal chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Dmem media, by Thermo Fisher Scientific (1 mentions) Chloroform, by Merck Group (1 mentions) Methanol, by Merck Group (1 mentions) Ammonium formate, by Merck Group (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Sodium dodecyl sulfate (sds), by Merck Group (1 mentions) Ammonium bicarbonate, by Merck Group (1 mentions) Asp n, by Promega (1 mentions)

20 protocols using high capacity streptavidin agarose beads

Quantitative Proteomic Analysis of Cell Signaling

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Iodoacetamide was purchased from GE Healthcare. RPMI-1640 and DMEM media were from Gibco. Microcon YM-30 spin filters were from Millipore. MS-grade Trypsin, Asp-N, and Arg-C were from Promega. Tris, SDS, methanol, chloroform, 2,2′-dithiodipyridine (DTDP), dime-thylformamide (DMF), hydroxylamine, sodium chloride, ammonium bicarbonate, and ammonium formate were from Sigma-Aldrich. Arginine (Arg0), lysine (Lys0), ¹³C₆¹⁵N₄-arginine (Arg10), ¹³C₆¹⁵N₂-lysine (Lys8), dialyzed fetal bovine serum, RPMI 1640 medium for SILAC, 660 nm Protein Assay Kit, TCEP, NEM, high-capacity streptavidin agarose beads, SuperSignal chemiluminescent substrate, trap columns, EASY-Spray analytical columns, and Horseradish peroxidase (HRP)-conjugated streptavidin were from Thermo Fisher Scientific. Primary antibodies were from Cell Signaling Technology, Novus Biologicals, Santa Cruz Biotechnology, Sigma-Aldrich, and R&D Systems. HRP-conjugated species-specific secondary antibodies were from Cell Signaling Technology and Jackson ImmunoResearch Laboratory.

Zhou B., Wang Y., Yan Y., Mariscal J., Di Vizio D., Freeman M.R, & Yang W. (2019). Low-Background Acyl-Biotinyl Exchange Largely Eliminates the Coisolation of Non-S-Acylated Proteins and Enables Deep S-Acylproteomic Analysis. Analytical chemistry, 91(15), 9858-9866.

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Membrane Fraction Analysis of TRPC1

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To determine the membrane fraction of TRPC1, 1 × 10⁶ HPNE cells or 8 × 10⁵ PANC-1 cells grown in normal pH, acid adaptation, or acid recovery conditions were seeded in 60 mm Petri dishes for 48 h and collected as previously described [45 (link)]. Briefly, cells were washed three times with cold PBS, then incubated with 3 mg of sulfo-NHS-SS-biotin (Thermo Fisher Scientific) and slightly shaken for 1 h at 4 °C. The reaction was interrupted by the addition of cold PBS containing 10 mM glycine. Cells were scrapped with RIPA buffer, and ~10% of the total lysate was saved as the total lysate fraction. The remaining lysate portion (corresponding to the membrane fraction) was incubated with high-capacity streptavidin agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) with gentle rotation overnight at 4 °C. After incubation, beads were washed four times with RIPA buffer. Proteins were eluted from the beads with 50 µL of Laemmli buffer 2X and heated at 60 °C for 30 min. Both total lysate samples and membrane fraction samples were used for Western blot analysis, as described above.

Schnipper J., Kouba S., Hague F., Girault A., Telliez M.S., Guénin S., Ahidouch A., Pedersen S.F, & Ouadid-Ahidouch H. (2022). Acid Adaptation Promotes TRPC1 Plasma Membrane Localization Leading to Pancreatic Ductal Adenocarcinoma Cell Proliferation and Migration through Ca2+ Entry and Interaction with PI3K/CaM. Cancers, 14(19), 4946.

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Enrichment of Biotinylated Proteins

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Samples after protein precipitation were centrifuged at 6000g for 5 min at 4°C. The supernatant was discarded, and the pellet was washed with ice-cooled methanol twice and air-dried for 10 min. The protein pellet was then dissolved in 1× PBS with 4% SDS, 20 mM EDTA, and 10% glycerol by vortexing and heating. The solution was then diluted with 1× PBS to give a final concentration of SDS as 0.5%. High-capacity streptavidin agarose beads (Thermo Fisher Scientific) were added to bind the biotinylated proteins with rotating for 1.5 hours at room temperature. The beads were washed stepwise with 1× PBS with 0.2% SDS, 6 M urea in PBS with 0.1% SDS, and 250 mM NH₄HCO₃ with 0.05% SDS (23 (link)).

Liu Z., Wu Y., Mao X., Kwan K.C., Cheng X., Li X., Jing Y, & Li X.D. (2023). Development of multifunctional synthetic nucleosomes to interrogate chromatin-mediated protein interactions. Science Advances, 9(18), eade5186.

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Affinity Capture of Protein Complexes

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PU-WS13-biotin beads were prepared by incubating 20 mM PU-WS13-biotin (chemical bait) with High Capacity Streptavidin agarose beads (ThermoFisher Scientific) for 3 hr at 4°C followed by washing with Felts buffer for three times. Antibody beads were prepared by incubating 9G10 or G4420 anti-GRP94 antibodies with protein A/G agarose beads (Roche) for 2 hr at 4°C followed by washing with Felts buffer for three times. The pre-formed chemical bait or antibody bait was then added into the cell lysate and the mixture was incubated on a rotator for 3 hr at 4°C. After separating the beads by centrifugation, the supernatant was collected and incubated with new pre-formed chemical bait or antibody bait. The sequential capture experiment was carried out by repeating the chemical precipitation (CP)/immunoprecipitation (IP) three times before the final IP with the indicated antibody bait. Captured cargos at each step were washed with Felts buffer three times before loading onto SDS-PAGE and subjecting to immunoblotting.

Yan P., Patel H.J., Sharma S., Corben A., Wang T., Panchal P., Yang C., Sun W., Araujo T.L., Rodina A., Joshi S., Robzyk K., Gandu S., White J.R., de Stanchina E., Modi S., Janjigian Y.Y., Hill E.G., Liu B., Erdjument-Bromage H., Neubert T.A., Que N.L., Li Z., Gewirth D.T., Taldone T, & Chiosis G. (2020). Molecular Stressors Engender Protein Connectivity Dysfunction through Aberrant N-Glycosylation of a Chaperone. Cell reports, 31(13), 107840.

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Affinity Purification of GRP94 Complexes

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Protein extracts were prepared in the indicated buffers and diluted in Felts buffer. Samples were incubated with D-biotin (Control), Inactive-WS13-biotin (Control), PU-WS13-biotin (GRP94 bait) or GRP94 antibodies for 3 hr at 4°C, followed by incubation with High Capacity Streptavidin agarose beads (ThermoFisher Scientific) or Protein A/G agarose beads (Roche) for another 2 hr at 4°C. The beads were washed with cold Felts buffer three times and subjected to immunoblotting.

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Detecting Cysteine Sulfenic Acid with DCP-Bio1

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Cysteine sulfenic acid detection by DCP-Bio1 probe was performed^{50 (link)}. The treated cells were washed with PBS and homogenized in DCP-Bio1 working lysis buffer (1× PBS, 1% NP40, 1% sodium deoxycholate, 5 mM EDTA, 5 mM EGTA, 1 mM PMSF, 0.1 mM aprotinin, 0.1 mM leupeptin, 10 mM NEM, 10 mM iodoacetamide, 100 µM DTPA, and 200 U mL⁻¹ catalase). Lysates were centrifuged at 12,000 × g for 10 min. Supernatants were collected and incubated with 1 mM DCP-Bio1 probe for 2 h on ice. For affinity capture of the labeled proteins, unreacted DCP-Bio1 was removed using a dialysis device. Dialyzed Samples were precleared with Sepharose CL-4B beads (Sigma), applied to plugged columns containing high-capacity streptavidin-agarose beads (ThermoFisher Scientific) and incubated overnight at 4 °C. Beads were then subjected to a series of stringent washes (at least four column volumes and two washes each) using 1% sodium dodecyl sulfate (SDS), 4 M urea, 1 M NaCl, 10 mM DTT, 100 μM ammonium bicarbonate and water. Beads were boiled for 10 min and samples were analyzed by immunoblotting analysis.

Zhou Z.S., Li M.X., Liu J., Jiao H., Xia J.M., Shi X.J., Zhao H., Chu L., Liu J., Qi W., Luo J, & Song B.L. (2020). Competitive oxidation and ubiquitylation on the evolutionarily conserved cysteine confer tissue-specific stabilization of Insig-2. Nature Communications, 11, 379.

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Purification of Proteasomes from Cell Lines

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Non-treated and DR-treated K562 cells were lysed for 40 min at 4 °C in a buffer containing 50 mM Trise-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS, 1x protease inhibitor cocktail (Roche). Proteasomes were extracted and purified from cytosol using a multistep purification procedure as described previously [56 (link)].
HEK293 cells with stable expression of Rpn11-HTBH were lysed for 30 min at 4 °C in 50 mM Trise-HCl, pH 7.5, 100 mM NaCl, 10% glycerol, 5 mM ATP, 1 mM DTT, 5 mM MgCl₂, 1x protease inhibitor cocktail (Roche), and 0.5% NP-40. Cell lysates were incubated with high-capacity streptavidin agarose beads (Thermo Scientific) overnight at 4°C. Streptavidin-immobilized proteasomes were purified as described previously[55 (link)]. For each purification batch, 10 μg of purified proteasomes were analyzed for purity on 12% SDS-PAG.

Tsimokha A.S., Kulichkova V.A., Karpova E.V., Zaykova J.J., Aksenov N.D., Vasilishina A.A., Kropotov A.V., Antonov A, & Barlev N.A. (2014). DNA damage modulates interactions between microRNAs and the 26S proteasome. Oncotarget, 5(11), 3555-3567.

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Quantification of FakB1 Binding Liposomes

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Five microliters of purified FakB1 (40 μM) was added to 20 μl of high-capacity streptavidin agarose beads (Thermo Fisher) and 25 μl of liposomes containing the following: 1 mol percent of DOPE-N-(cap biotinyl) and 99 mol percent of DOPC or DOPG; 1 mol percent of DOPE-N-(cap biotinyl), 89 mol percent DOPG, and 10 mol percent of [¹⁴C]16:0; 1 mol percent of DOPE-N-(cap biotinyl), 79 mol percent DOPG, and 20 mol percent of [¹⁴C]16:0. Buffer (0.1 M Tris-HCl, pH 7.5) was added to a total volume of 200 μl, and samples were incubated at room temperature on a shaker for 1 h before being centrifuged at 2000g for 5 min. Supernatant was decanted without disturbing the streptavidin beads, and excess moisture was wicked from the beads before resuspending in an equal volume of buffer. A Western blot was performed on aliquots from the supernatant and pellet using monoclonal anti-polyHistidine−alkaline phosphatase antibody (Sigma). Blots were images on a Typhoon FLA 9500, and band intensities were quantified using ImageQuant (Cytiva). Statistical significance was determined using the two-tailed Student’s t test.

Gullett J.M., Cuypers M.G., Grace C.R., Pant S., Subramanian C., Tajkhorshid E., Rock C.O, & White S.W. (2022). Identification of structural transitions in bacterial fatty acid binding proteins that permit ligand entry and exit at membranes. The Journal of Biological Chemistry, 298(3), 101676.

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Affinity Purification of YK5-Biotin Complexes

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Protein lysates were prepared using 20 mM Tris pH 7.4, 25 mM NaCl, 0.1% NP-40 lysis buffer. High capacity streptavidin agarose beads (50 μL) (Thermo Scientific) were washed three times with the lysis buffer. The indicated concentration of YK5-biotin was added to the streptavidin beads, and the mixture was incubated at 4 °C for 1 h. Upon a three-time wash with the buffer, YK5-biotin/SBs were added to 500 μg aliquots of total cellular protein in the lysis buffer. Samples were incubated at 4 °C overnight, washed five times with the lysis buffer, and applied to SDS-PAGE. Gels were either subjected to Western blotting procedure or stained by Coomassie blue (Bio-Rad) or silver (Invitrogen) as indicated.

Rodina A., Taldone T., Kang Y., Patel P.D., Koren J I.I.I., Yan P., DaGama Gomes E.M., Yang C., Patel M.R., Shrestha L., Ochiana S.O., Santarossa C., Maharaj R., Gozman A., Cox M.B., Erdjument-Bromage H., Hendrickson R.C., Cerchietti L., Melnick A., Guzman M.L, & Chiosis G. (2014). Affinity Purification Probes of Potential Use To Investigate the Endogenous Hsp70 Interactome in Cancer. ACS Chemical Biology, 9(8), 1698-1705.

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Evaluating Biotin Compounds Binding to r(CGG)12 RNA

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The r(CGG)₁₂, 5′-GCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGC, was 5′-end-labeled with ³²P using [γ-³²P] ATP. To determine if 2H-5-CA-Biotin, 2P-5-CA-Biotin, 2H-5-MA-Biotin, and 2P-5-MA-Biotin react with r(CGG)₁₂in vitro, 5 μL of ³²P-labeled r(CGG)₁₂ (∼1,000,000 cpm) was diluted in a total volume of 300 μL of 1× PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 137 mM NaCl, and 2.7 mM KCl, pH 7.4). The RNA was folded by heating at 60 °C for 5 min and slowly cooling to room temperature. Compound was then added at various concentrations, and the solutions were incubated for at least 4 h at room temperature. Next, 10 μL of streptavidin resin (high capacity streptavidin agarose beads; Thermo Scientific) was added to the samples which were then incubated for 30 min at room temperature. After centrifugation, the supernatant was removed, and the resin was washed with 1× PBST (1× PBS + 0.1% Tween 20). The amount of radioactivity in the supernatant, wash, and associated with the beads was measured using a Beckman Coulter LS6500 liquid scintillation counter.

Yang W.Y., Wilson H.D., Velagapudi S.P, & Disney M.D. (2015). Inhibition of Non-ATG Translational Events in Cells via Covalent Small Molecules Targeting RNA. Journal of the American Chemical Society, 137(16), 5336-5345.

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