Dynabeads

Manufactured by New England Biolabs

Dynabeads are magnetic beads used for a variety of biomolecule isolation and purification applications. They are made of a polymer matrix with embedded magnetic particles, allowing for rapid and efficient separation using a magnetic field. Dynabeads are available in different sizes and surface chemistries to suit a wide range of biomolecular targets, including proteins, nucleic acids, and cells.

Automatically generated - may contain errors

Lab products found in correlation

6 protocols using dynabeads

Affinity Purification and Mass Spectrometry of 3XFLAG-GLD4-myc Protein Complexes

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

3XFLAG-GLD4-myc sequence was subcloned from p3xFLAG-myc CMV26 GLD4 (Burns et al. 2011 (link)) and inserted into pTRIPZ vector using AgeI and XhoI sites. U87MG cells were transduced with inducible pTRIPZ lentivirus expressing RFP (control) or 3XFLAG-GLD4-myc, and the expression was induced with 1 µg/mL doxycycline for 48 h. Freshly lysed cells were incubated with FLAG antibody conjugated Dynabeads G (Life Technologies), washed and eluted with 3X FLAG peptides (Sigma) followed by a second round of coimmunoprecipitation with myc antibody conjugated Dynabeads (NEB). Affinity purified protein complexes were electrophoresed a short distance into a nondenaturing gel. After in-gel digest, the peptides were run on the QExactive (Thermo Scientific) LC–MS/MS at the proteomics and mass spectrometry facility at the University of Massachusetts Medical School. Data were analyzed with an intensity-based absolute quantification method, and differential protein abundance was calculated as fold change relative to the control using Scaffold software (Proteome Software).

Shin J., Paek K.Y., Chikhaoui L., Jung S., Ponny S., Suzuki Y., Padmanabhan K, & Richter J.D. (2022). Oppositional poly(A) tail length regulation by FMRP and CPEB1. RNA, 28(5), 756-765.

+ Open protocol

+ Expand

In Situ Hi-C Protocol for Chromatin Conformation Capture

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

In situ Hi-C was performed as previously described^{9 (link)} with slight modifications. Cells at 80% confluence in 10 cm dish were fixed in 1% pFA at room temperature for 10 min and subjected to the in situ Hi-C procedure. After proximal ligation, ligation products were enriched by centrifugation at 10,000 rpm at 4 °C for 10 min. The pellet was suspended with proteinase K solution consisting of 10 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 2 mg/ml proteinase K (Thermo Fisher Scientific) and incubated at 37 °C for 1 h, followed by overnight incubation at 68 °C with 0.5 M NaCl. DNA was purified by phenol/chloroform extraction and sonicated at 4 °C using Bioruptor (Diagenode) for 15 min by repeating the cycle of 30-sec ON and 1-min OFF. Biotin-labeled DNA was purified using Dynabeads (MyOne Streptavidin T1, Thermo Fisher Scientific). Sequencing adapters were ligated using NEBNext Ultra Ligation module (New England Biolabs, Inc) on Dynabeads, and DNA was PCR-amplified for 8–14 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs, Inc). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp paired-end reads.

Iwasaki O., Tanizawa H., Kim K.D., Kossenkov A., Nacarelli T., Tashiro S., Majumdar S., Showe L.C., Zhang R, & Noma K.I. (2019). Involvement of condensin in cellular senescence through gene regulation and compartmental reorganization. Nature Communications, 10, 5688.

+ Open protocol

+ Expand

PRO-seq protocol with modifications

Check if the same lab product or an alternative is used in the 5 most similar protocols

We performed PRO-seq following a previously described protocol^{50 (link)} with some modifications. Nuclei were isolated by Dounce homogenizer with loose pestle. Approximately 10⁷ nuclei were subjected to nuclear run-on (30 °C, 3 min) in the presence of 25 μM biotin-11-ATP/GTP/CTP/UTP (PerkinElmer) and 6 × 10⁵Drosophila S2 spike-in nuclei. Total RNAs were extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). Biotinylated nascent RNA was purified by Dynabeads M-280 streptavidin (Invitrogen). After 3′ VRA3 adapter ligation and the second purification by Dynabeads, 5′ cap and 5′ hydroxyl RNA were converted to 5′ monophosphorylated RNA by RppH (NEB) and PNK (NEB), respectively. After 5′ VRA5 adapter ligation and the third purification by Dynabeads, complementary DNA was generated by reverse transcription with SuperScript III (Invitrogen) and RP1 primer. PCR by Phusion Hot Start (Thermo) and RP1/RPIx primer sets in 10 cycles amplified indexed DNA libraries. DNA libraries of 140–350 bp were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science). Two independent cell cultures per condition were then sequenced by NextSeq 500 system (Illumina) with single-read runs.

Douillet D., Sze C.C., Ryan C., Piunti A., Shah A.P., Ugarenko M., Marshall S.A., Rendleman E.J., Zha D., Helmin K.A., Zhao Z., Cao K., Morgan M.A., Singer B.D., Bartom E., Smith E.R, & Shilatifard A. (2020). Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb, and DNA methylation. Nature genetics, 52(6), 615-625.

+ Open protocol

+ Expand

Identifying eIF3 Subunits by PAR-CLIP

Check if the same lab product or an alternative is used in the 5 most similar protocols

To identify eIF3 subunits that crosslinked with RNAs in the PAR-CLIP experiments, a portion of eIF3 immunoprecipitated using Dynabeads as described above were treated with nonradioactive ATP (NEB) during the T4 polynucleotide kinase labeling step. The nonradioactive samples were then run on the same gel next to the radiolabeled PAR-CLIP samples, Coomassie stained (Pierce) and the bands that matched with the phosphorimager printout were excised from the gel and submitted for identification using one-dimensional LC-MS/MS (Supplementary file 1).

De Silva D., Ferguson L., Chin G.H., Smith B.E., Apathy R.A., Roth T.L., Blaeschke F., Kudla M., Marson A., Ingolia N.T, & Cate J.H. (2021). Robust T cell activation requires an eIF3-driven burst in T cell receptor translation. eLife, 10, e74272.

+ Open protocol

+ Expand

Profiling Translatome Changes in Endoplasmic Reticulum Stress

Check if the same lab product or an alternative is used in the 5 most similar protocols

Briefly, HepG2 cells were transfected with pSicoR-eIF1a-GFP-RPL10a. 48 hours after transfection, the cells were stimulated with Tm (1ug/ml) for 8 hours. 10min before cell harvest, 100 μg/mL of CHX was added in culture media for 10 min. Cells were homogenized in ice-cold lysis buffer (20 mM HEPES, 5 mM MgCl2, 150 mM KCl, 1% NP-40, 0.5 mM DTT, 100 mg/ml cyclohexamide, RNase inhibitors and protease inhibitors) by passing through pre-chilled 23- then 25-gauge needles 10× each. Separate 10% of the lysate for input of the qPCR and 90% of samples centrifuged at 2,000 x g for 10 min to remove large debris. Supernatants were incubated with streptavidin/protein G-coated Dynabeads (New England Biolabs) bound to anti-GFP antibodies for 1 hours at 4 °C with gentle mixing. Anti-GFP beads were washed with high salt buffer (20 mM HEPES, 5 mM MgCl2, 350 mM KCl, 1% NP-40, 0.5 mM DTT and 100 mg/ml cyclohexamide) and RNA was eluted from all samples using RNeasy Kits (Qiagen) according to the manufacturer’s instructions. RNA yield was quantified using RiboGreen (Life Technologies) and RNA quality was determined by Bioanalyzer analysis.

Wei J., Harada B.T., Lu D., Ma R., Gao B., Xu Y., Montauti E., Mani N., Chaudhuri S., Gregory S., Weinberg S., Zhang D., Green R., He C, & Fang D. (2021). HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease. Molecular cell, 81(24), 5052-5065.e6.

+ Open protocol

+ Expand

Gα Subunit Peptide Synthesis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Ascorbic acid, (−)-Isoproterenol (+)-bitartrate salt and clenbuterol hydrochloride were purchased from MilliporeSigma. Polyethyleneimine (PEI) 25 kDa linear polymer was purchased from PolySciences, Streptavidin-coated magnetic beads (Dynabeads) were purchased from New England Biolabs. Ni-NTA agarose was purchased from Qiagen. Synthetic peptides of the C-terminal α5 helix of the Gα subunit were synthesized at >95% purity from Genscript. Lyophilized peptides were dissolved in water and the concentration determined by the mass of the lyophilized peptide. Biotinylated Spep was synthesized at >95% purity from Genscript with an N-terminal biotin conjugation.

Culhane K.J., Gupte T.M., Madhugiri I., Gadgil C.J, & Sivaramakrishnan S. (2022). Kinetic model of GPCR-G protein interactions reveals allokairic modulation of signaling. Nature Communications, 13, 1202.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!