For histone peptide pull-down assay, 1.5 μg of biotinylated histone peptides were incubated with streptavidin beads (NEB) in binding buffer (50 mM Tris-HCl 8.0, 300 mM NaCl, and 0.1% NP-40) for 1 h at 4 °C, and then washed with binding buffer. A 1.5 μg quantity of AIPP3-BAH proteins was incubated with a peptide–bead mixture in 0.5 ml of binding buffer for 3 h at 4 °C. and then washed with binding buffer five times. The protein–bead mixtures were subjected to immunoblotting using anti-GST antibody (Abmart, #12G8, 1:2000 dilution).
Streptavidin beads
Manufactured by New England Biolabs
Sourced in United States
Streptavidin beads are a type of magnetic bead coated with the protein streptavidin. Streptavidin has a high affinity for biotin, a small molecule commonly used to label proteins, nucleic acids, and other biomolecules. The streptavidin-coated beads can be used to capture and isolate biotinylated targets from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti gst antibody, by Abmart (2 mentions)
α flag, by Merck Group (2 mentions)
α gapdh, by Santa Cruz Biotechnology (2 mentions)
17β estradiol e2, by Merck Group (2 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Fulvestrant from, by MedChemExpress (2 mentions)
Rnase a t, by Thermo Fisher Scientific (2 mentions)
αkdm2a, by Proteintech (2 mentions)
αpol 2, by Santa Cruz Biotechnology (2 mentions)
αrad21, by Abcam (2 mentions)
αlamina c, by Abcam (2 mentions)
Hispur cobalt superflow agarose beads, by Thermo Fisher Scientific (2 mentions)
Rnase t1 cocktail, by Thermo Fisher Scientific (2 mentions)
Direct zol rna kit, by Zymo Research (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Agencourt cleanseq beads, by Beckman Coulter (1 mentions)
Bio gel p 2, by Bio-Rad (1 mentions)
8 protocols using streptavidin beads
1
Histone Peptide Pull-Down Assay
2
Antibodies and Reagents for Epigenetic Studies
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Antibodies used in this study include: αERα (Bethyl #A300–495A) for ChIP-qPCR, ChIP-Seq, RIP-qPCR, PAR-CLIP and immunoblotting; αKDM2A (ProteinTech #24311–1-AP) for ChIP-qPCR; αPol II (Santa Cruz Biotechnology #SC-9001, #SC-56767 and Abcam #ab817) for ChIP-qPCR; αRAD21 (Abcam #ab992) for ChIP-qPCR. Other antibodies that were applied in western blot include: αGST (ProteinTech #66001–2); αHis (ProteinTech #66005–1); αHA (BioLegend, #901501); αNEDD4 (CST #3607); αPol II (Santa Cruz Biotechnology #SC-56767), αLaminA/C (Abcam, # ab8984), αGAPDH (Santa Cruz Biotechnology #sc-365062) and αFLAG (Sigma #F1804). Reagents that were utilized for immunoprecipitation purposes are: anti-HA magnetic beads (Thermo Fisher #88837); streptavidin beads (NEB #S1420S); HisPurTM cobalt Superflow agarose beads (Thermo Fisher #25228); Dynabeads Protein A (Thermo Fisher #10006D) or Protein G (Thermo Fisher #10007D). Normal anti-rabbit IgG antibody was purchased from Invitrogen (#02–6102), 17β-estradiol (E2) from Sigma (#E2758), fulvestrant from Medchem Express (#HY13636), RNase A/T from Thermo Fisher (#EN0551), RNase/T1 cocktail from Thermo Fisher (#AM2286) and iTaq Universal SYBR Green Supermix from BioRad (1725124). Antibodies information can also be found in Key Resources Table .
3
Antibodies and Reagents for Epigenetic Studies
4
Nucleosome Assembly and Immobilization
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Biotinylated GBY and 147 bp DNA was prepared as previously described [33 (link)]. Briefly, recombinant Xenopus laevis histones were prepared [34 (link)] and assembled into octamers [35 (link)]. Nucleosomes were assembled by depositing the histone octamer onto DNA by the process of rapid dilution and then dialyzing into LDB buffer (2.5 mM NaCl, 10 mM Tris pH 7.4, 0.25 mM EDTA), as previously described [36 (link)]. The homogeneity and degree of saturation of nucleosome assemblies were assessed via 4% Native PAGE. Nucleosomes were immobilized on hydrophilic streptavidin beads from NEB as previously described [37 (link)].
5
Cusativin Purification from E. coli
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Streptavidin beads and Proteinase K were purchased from New England Biolab (NEB), Direct-zol RNA Kit from Zymo Research. Ribonuclease T1 was procured from Worthington Biochemical Corporation. Cusativin was expressed and purified from E. coli based over an expression system as described before (Addepalli et al. 2017 (link)). All other chemicals were procured from Fisher Scientific, unless specified.
6
Viral sfRNA Enrichment and Sequencing
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Total RNA was extracted from WT and ΔsfRNA stable cell lines with Trizol (Invitrogen Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). The isolated RNA was then mixed with biotin-modified probes specific for WNV 3′ UTR (BIO-WNRTRe, BIO-WNRTReb) in order to capture the viral sfRNA species. The probes are listed on Table S1 . After that, the sfRNA–probe complexes were immobilized on streptavidin beads (New England Biolabs, Ipswich, MA, USA), and the unwanted cellular RNA was removed. These sfRNA-enriched samples were then sequenced.
7
Characterization of SARS-CoV-2 RBD Glycans
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RBD (BEI NR-52309) was biotinylated with sulfosuccinimidyl-6-[biotinamido]-6-hexanamido hexanoate (ThermoScientific) and coupled to streptavidin beads (New England Biolabs). Sera were incubated with RBD-coupled beads. RBD-specific antibodies were eluted using 100 mM citric acid (pH 3.0) and neutralized with 0.5 M potassium phosphate (pH 9.0). IgG was isolated by protein G (Millipore) and deglycosylated using PNGase (New England Biolabs). Glycans were labeled with 8-aminoinopyrene-13,6-trisulfonic acid (ThermoFisher), unbound removed using Agencourt CleanSEQ beads (Beckman Coulter) (all IgG glycans) and Bio-Gel P-2(Bio-rad) (RBD-specific glycans), quantitated using ABI 3500xL, and analyzed with GlycanAssure version 1.0 [8 (link), 35 (link), 36 (link)].
8
Histone Peptide Pull-Down Assay
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Histone peptide pull-down was performed according to a previous report 27 . In brief, 1.5 μg of biotinylated histone peptides were incubated with streptavidin beads (NEB) in binding buffer (50 mM Tris-HCl 8.0, 300 mM NaCl, 0.1% NP-40) for 1 h at 4 °C and then washed with binding buffer. A 1.5 μg quantity of AIPP3-BAH proteins was incubated with a peptide-bead mixture in 0.5 ml of binding buffer for 3 h at 4 °C and then washed with binding buffer five times. The protein-bead mixtures were subjected to immunoblotting using anti-GST antibody (Abmart).
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