Dynabeads

Manufactured by Merck Group

Sourced in United States

Dynabeads are a type of magnetic beads used in various laboratory applications. They are composed of a polymer matrix with embedded magnetic particles, allowing for magnetic separation and manipulation of target molecules or cells. Dynabeads are designed to provide a versatile platform for applications such as cell isolation, protein purification, and nucleic acid extraction, among others.

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Lab products found in correlation

Dynabead, by Thermo Fisher Scientific (8 mentions) Flag antibody, by Merck Group (3 mentions) Protein g dynabead, by Thermo Fisher Scientific (2 mentions) Anti flag m2 antibody, by Merck Group (2 mentions) Benzonase, by Merck Group (2 mentions) Easysep, by STEMCELL (1 mentions) Slx4ip, by Merck Group (1 mentions) Recombinant dnase 1, by Merck Group (1 mentions) Dynabeads co immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Complete protease inhibitor tablet, by Roche (1 mentions) French press, by Constant Systems (1 mentions) Phosphatase inhibitor tablet, by Roche (1 mentions) Anti rank, by R&D Systems (1 mentions) Trypsin, by Merck Group (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Rhodamine b, by Merck Group (1 mentions) Gfp antibody, by Merck Group (1 mentions) Halt proteinase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)

32 protocols using dynabeads

Preparation and Infection of CD4+ Lymphocytes

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CD4⁺ lymphocytes were prepared as follows; Adherence-depleted PBMC were depleted of CD56⁺ cells (Easy Sep, StemCell Technologies) and CD8⁺ cells (Dyna beads, Sigma) by negative selection. CD4⁺ lymphocytes were stimulated in R10 and phytohaemagglutinin (PHA, 5μg/ml). IL-2 (500 IU/ml) was added to the culture 24h following PHA stimulation. CD4⁺ T cells were infected with HIV-1 by spinoculation (2500rpm at 25°C for 2–3h) 48–72h following PHA stimulation, as described (Norman et al., 2011 (link)). Infected cells were maintained in R10 and IL-2 until analyzed.

Mashiba M., Collins D.R., Terry V.H, & Collins K.L. (2014). Vpr overcomes macrophage-specific restriction of HIV-1 Env expression and virion production. Cell host & microbe, 16(6), 722-735.

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SLX4IP Protein Interaction Analysis

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RNA was isolated, quantified, and analyzed as described previously.^{35 (link)} Protein isolation, western blotting analysis and quantification was completed as published previously.^{35 (link)} Primary antibody dilutions are as follows: 1:10,000 GAPDH (Cell Signaling, 2118), 1:3,000 SLX4IP (Sigma, HPA046372), 1:3,000 FLAG (Sigma, F1804), 1:5,000 TRF2 (Novus Biologicals, NB110–57130), 1:1,000 PML (Cell Signaling, E5R8T and Santa Cruz Biotechnology, sc-5621), 1:3,000 p21 (Cell Signaling, 2947).
Coimmunoprecipitation experiments were carried out using the Dynabeads™ Co-Immunoprecipitation Kit according to manufacturer instructions (ThermoFisher Scientific). The provided immunoprecipitation (IP) buffer included was modified with an additional 50 mM NaCl and 0.05% Triton X-100. Cells were grown in 15 cm dishes and scraped using the modified IP buffer, treated with 5 μL of recombinant DNaseI (Sigma), rotated at 4° C for one hour, and cleared by centrifugation. Following protein quantification, 2 mg of protein was incubated with 1.5 mg of Dynabeads coupled with 10 µL of FLAG antibody (Sigma, F1804) or 20 µL of SLX4IP antibody (Sigma, HPA046372) per protocol. After completion, whole cell lysates and coimmunoprecipitation samples were analyzed by western blotting as described above.

Mangosh T.L., Grabowska M.M, & Taylor D.J. (2021). SLX4IP N-terminus dictates telomeric localization in ALT-like castration-resistant prostate cancer cell lines. The Prostate, 81(15), 1235-1251.

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Affinity Purification of Atg18 Protein

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For Atg18 purification in SILAC experiments, pellets were thawed on ice in one pellet volume of TGN lysis buffer (50 mM Tris pH 7.4, 10% Glycerol, 100 mM NaCl) supplemented with 0.5% Triton, complete protease inhibitor tablets (Roche), phosphatase inhibitor tablets (Roche), 1 mM DTT, and 1 mM PMSF. Cells were passed one time through a French press (Constant Systems LTD) at 4°C with 2.2 kpsi of pressure. Atg18 was isolated by affinity purification using the Gly₆‐FLAG₃ tag and Dynabeads (Sigma) crosslinked with the FLAG M2 antibody (F1804 epitope, Sigma). After 1 h of incubation at 4°C, beads were washed three times with TGN buffer using a magnetic rack, transferred to new Eppendorf tubes, washed with 1 ml of elution buffer (Tris 50 mM pH 7.4), then eluted with 3xFLAG peptide (0.5 mg/ml) in elution buffer. Eluates were flashed‐frozen in liquid nitrogen and placed on a −80°C freezer before Coomassie staining and MS analysis.

Courtellemont T., De Leo M.G., Gopaldass N, & Mayer A. (2022). CROP: a retromer‐PROPPIN complex mediating membrane fission in the endo‐lysosomal system. The EMBO Journal, 41(10), e109646.

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Thymic Organoid Isolation and Analysis

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Embryonic day 15 lobes were cultured with 1.35 mM 2 deoxyguanosine (2dGuo) for 7 d (Cowan et al., 2013 (link)). 2dGuo FTOCs were stimulated with 2 µg/ml each of anti-RANK (R&D Systems) or anti-LTβR (Banks et al., 2005 (link)) for 4 d. Lobes were digested using 0.25% trypsin/0.02% EDTA (Sigma-Aldrich) and depleted of any remaining CD45⁺ with Dynabeads (Anderson et al., 1993 (link)). Cells were then snap frozen for quantitative PCR (qPCR) or permeabilized and stained with antibodies to CD45, EpCAM1, Ly51, Aire, and Fezf2.

Cosway E.J., Lucas B., James K.D., Parnell S.M., Carvalho-Gaspar M., White A.J., Tumanov A.V., Jenkinson W.E, & Anderson G. (2017). Redefining thymus medulla specialization for central tolerance. The Journal of Experimental Medicine, 214(11), 3183-3195.

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Quantifying Targeting Efficiency via Dynabeads

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To enable targeting efficiency quantification, 20 µL of 4.5 µm epoxy Dynabeads^® were incubated in 0.1 mg/mL rhodamine B (Sigma Aldrich) for 24 h. After incubation, the beads were washed five times with 0.2 wt% SDS solution. The bead solution was stored in darkness until used, and, if greater than 5 h elapsed, were washed 3× using the same process to remove any desorbed rhodamine B.

Zimmermann C.J., Herson P.S., Neeves K.B, & Marr D.W. (2022). Multimodal microwheel swarms for targeting in three-dimensional networks. Scientific Reports, 12, 5078.

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Double Co-Immunoprecipitation for Protein Complex Analysis

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Cells were grown to 90% confluence in 6-well or 60 mm plates and transfected with plasmids. After 24 h cellular proteins were cross-linked with 1 mM DSP (dithiobis(succinimidyl propionate), Thermo Scientific) in PBS. Reaction was stopped after 30 min by adding Tris-HCl pH 7,5 to the final concentration 20 mM. First co-IP was carried out as described above. Precipitated protein complexes were eluted from beads with 100 mM glicyne pH 2,5 and solution was immediately neutralized by adding Tris-HCl pH 8,0 to the final concentration 150 mM. Eluted proteins were diluted with IP buffer [79 (link)] and second Co-IP was performed as described above. Antibodies used for immunoprecipitation were cross-linked to Dynabeads with 20 mM DMP (dimethyl pimelimidate, Sigma-Aldrich) in 200 mM triethanolamine pH 8,2 (Sigma-Aldrich) for 45 min.

Tracz-Gaszewska Z., Klimczak M., Biecek P., Herok M., Kosinski M., Olszewski M.B., Czerwińska P., Wiech M., Wiznerowicz M., Zylicz A., Zylicz M, & Wawrzynow B. (2017). Molecular chaperones in the acquisition of cancer cell chemoresistance with mutated TP53 and MDM2 up-regulation. Oncotarget, 8(47), 82123-82143.

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Isolation of GFP-LC3 Autophagy Vesicles

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Cortex samples from 12-week old 5XFAD; Becn1^FA/FA; GFP-LC3 mice were dissected and homogenized in 1 ml cold lysis buffer pH 7.4 containing 250 mM sucrose, 1 mM EDTA, 10 mM HEPES, halt proteinase inhibitor cocktail (ThermoFisher Scientific), and halt phosphatase inhibitor cocktail (ThermoFisher Scientific), using a Dounce tissue grinder (Wheaton). The lysate was then passed 15 times through 27-gauge needle. GFP-based immunoisolation was performed using Dynabeads Protein G (ThermoFisher Scientific). The lysate was centrifuged at 1,000 x g for 10 min at 4°C. The post-nuclear supernatant fraction was centrifuged at 20,000 x g for 20 min and the supernatant fraction was discarded to remove residual cytosolic GFP-LC3 [61 (link)]. The pellet fraction was resuspended in 250 μl lysis buffer and was incubated for 2 hours at 4°C with 40 μl of Dynabeads, preincubated O/N with GFP-antibody (Sigma, G1544). The beads were then washed 4 times with wash buffer (150 mM NaCl, 250 mM sucrose, 1 mM EDTA, 10 mM HEPES) using the magnetic Separator DynaMag^TM-2 (ThermoFisher Scientific). Immunoprecipitates were eluted with lysis buffer containing 1X sample buffer and analyzed by SDS-PAGE.

Rocchi A., Yamamoto S., Ting T., Fan Y., Sadleir K., Wang Y., Zhang W., Huang S., Levine B., Vassar R, & He C. (2017). A Becn1 mutation mediates hyperactive autophagic sequestration of amyloid oligomers and improved cognition in Alzheimer's disease. PLoS Genetics, 13(8), e1006962.

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NLRP3 Inflammasome Regulation by Kinase Inhibitors

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HEK293T were transfected using CaPO₄ and 24 h later, treated with 1 μM MCC950 or 60 μM acalabrutinib for 6 h or with 60 μM ibrutinib for 2 h followed by 4-h incubation with medium without ibrutinib, where indicated. Cells were lysed 48 h after transfection in RIPA buffer supplemented with protease/phosphatase inhibitors (Roche). Lysates were subjected to immunoprecipitation of the NLRP3-HA or NACHT-FLAG fusion protein with Dynabeads (Sigma-Aldrich) or with agarose beads covered with PI4P (P-B004a; Echelon Biosciences). Washed beads were boiled in loading buffer and applied to standard SDS-PAGE on Thermo Fisher Scientific precast gels, followed by immunoblot according to the antibody manufacturer’s instructions. Membranes were exposed using Fusion FL camera and FusionCapt Advance software (PEQLAB). Quantification was conducted using the same software.

Bittner Z.A., Liu X., Mateo Tortola M., Tapia-Abellán A., Shankar S., Andreeva L., Mangan M., Spalinger M., Kalbacher H., Düwell P., Lovotti M., Bosch K., Dickhöfer S., Marcu A., Stevanović S., Herster F., Cardona Gloria Y., Chang T.H., Bork F., Greve C.L., Löffler M.W., Wolz O.O., Schilling N.A., Kümmerle-Deschner J.B., Wagner S., Delor A., Grimbacher B., Hantschel O., Scharl M., Wu H., Latz E, & Weber A.N. (2021). BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity. The Journal of Experimental Medicine, 218(11), e20201656.

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Circularized RNA Immunoprecipitation Assay

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circRIP assay was performed using the protocol from GeneSeed. Briefly, cells were sonicated after fixation with formaldehyde (Sigma). Then, the supernatant was incubated with the biotinylated circNUP98 or control probe (RioBio) and the magnetic streptavidin Dynabeads (Sigma). After total RNA extraction, the enrichment was measured by qRT‐PCR.

Yu R., Yao J, & Ren Y. (2020). A novel circRNA, circNUP98, a potential biomarker, acted as an oncogene via the miR‐567/ PRDX3 axis in renal cell carcinoma. Journal of Cellular and Molecular Medicine, 24(17), 10177-10188.

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Antibody-Conjugated Magnetic Bead Protocol

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Protein G Dynabeads (500 μL; Invitrogen) were washed twice in 1 mL 0.1M Na-phosphate, pH 7.0, before incubating with 50 μL M2 anti-FLAG monoclonal antibody (SIGMA) and 50 μL of 0.1M Na-phosphate with gentle agitation for 30 min at room temperature. Beads were washed twice in 1 mL of 0.1 M Na-phosphate pH 7.0 with 0.01% Tween 20, then washed twice with 1 mL of 0.2 M triethanolamine, pH 8.2. Antibody-conjugated Dynabeads were resuspended in 1 mL of 20 mM DMP (Dimethyl Pimelimidate, D8388, Sigma) in 0.2 M triethanolamine, pH 8.2 (prepared immediately before use) and incubated with rotational mixing for 30 min at room temperature. Beads were concentrated, the supernatant removed and 1 mL of 50 mM Tris-HCl, pH 7.5 added before incubating for 15 min with rotational mixing. The supernatant was removed and beads were washed three times with 1 mL 1XPBST+0.1% Tween-20 before resuspending in 300 mL of 1xPBST.

Borek W.E., Vincenten N., Duro E., Makrantoni V., Spanos C., Sarangapani K.K., de Lima Alves F., Kelly D.A., Asbury C.L., Rappsilber J, & Marston A.L. (2021). The Proteomic Landscape of Centromeric Chromatin Reveals an Essential Role for the Ctf19CCAN Complex in Meiotic Kinetochore Assembly. Current Biology, 31(2), 283-296.e7.

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