Hitrap streptavidin hp column

Manufactured by GE Healthcare

The HiTrap Streptavidin HP column is a pre-packed chromatography column used for the purification of biotinylated molecules. The column contains streptavidin, a protein that binds strongly to biotin, allowing for the capture and purification of biotinylated proteins, peptides, nucleic acids, or other biomolecules from complex samples.

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Lab products found in correlation

Hitrap, by Cytiva (4 mentions) Taq polymerase, by New England Biolabs (1 mentions) Profinia purification system, by Bio-Rad (1 mentions) Akta pure, by GE Healthcare (1 mentions) Hiload 16 600 superdex 200 pg gel filtration column, by GE Healthcare (1 mentions)

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HiTrap HP column (20 citations) HiTrap columns (11 citations) Streptavidin column (2 citations)

5 protocols using hitrap streptavidin hp column

Preparation of DNA Fragments for Biophysical Studies

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Two DNA fragments of 1440 and 609 bp were amplified from pBR322 plasmid by PCR using Taq polymerase (NEB) and the pairs of primers Cy5-2574⁺ and biotin-4014⁻, biotin-2574⁺ and 3185⁻, respectively. The biotinylated PCR products were purified on a MiniQ 4.6/50 ion exchange column (GE Healthcare Life Sciences) and loaded onto a HiTrap Streptavidin HP column (GE Healthcare Life Sciences). Purification of the nonbiotinylated strand was achieved by elution with 80 mM NaOH, neutralized by addition of HCl 1 M and annealed at equimolar concentrations in molecules, in presence of 1.5 mM MgCl₂ then purified on anion exchange MiniQ column.
For the ds–ss–ds, in this article, mentioned as gap substrate, three DNA fragments of 400, 600, and 1440 bp were amplified from pBR322 plasmid by PCR using the pairs of primers biotin-2574⁺ and Cy5-2974⁻ (400 bp), biotin-3413⁺ and Cy5-4014⁻ (600 bp), and 2574⁺ and biotin-4014⁻ (1440 bp). The three nonbiotinylated ssDNA fragments were purified by elution on HiTrap Streptavidin HP column and annealed at equimolar concentrations in molecules as described then purified on the anion exchange MiniQ column mentioned above.

Benureau Y., Moreira Tavares E., Muhammad A.A., Baconnais S., Le Cam E, & Dupaigne P. (2020). Method combining BAC film and positive staining for the characterization of DNA intermediates by dark-field electron microscopy. Biology Methods & Protocols, 5(1), bpaa012.

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Extraction and Analysis of tRNA Asp GUC

Cited 1 time

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Bulk tRNA was prepared from fresh cells from 750
mL of cultures of the different S. pombe strains
grown in YPD (1% yeast extract, 2% peptone, 2% dextrose, BD Difco
YPD) or 0.5% yeast extract (Difco), 2% bactopeptone (Difco), and 3%
dextrose (Fluka) at 30 °C, as described previously.^{85 (link)} tRNA^Asp_GUC was extracted
from bulk tRNA using a biotinylated primer (5′biotin-GCAAGCGTGACAGGCTTG-3′,
Integrated DNA Technologies) bound to a HiTrap Streptavidin HP column
(1 mL, GE Healthcare Life Sciences).^{85 (link)} Twenty-five
micrograms of tRNA^Asp_GUC was digested to nucleosides
that were then separated by LC–MS.^{85 (link)} To compare tRNA concentrations, we compared the ratio of the levels
of the modified bases Q (410 m/z) and m⁵C (258 m/z)
in each sample by integrating the peak area from the extracted ion
chromatograms. All tRNA extractions and analysis for Q content were
performed at least twice independently.

Zallot R., Brochier-Armanet C., Gaston K.W., Forouhar F., Limbach P.A., Hunt J.F, & de Crécy-Lagard V. (2014). Plant, Animal, and Fungal Micronutrient Queuosine Is Salvaged by Members of the DUF2419 Protein Family. ACS Chemical Biology, 9(8), 1812-1825.

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Purification and Characterization of Egg Yolk IgY

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Approximately 385 nmol of Y4‐4 peptide was immobilized on a HiTrap Streptavidin HP column (1 mL; GE Healthcare), according to the manufacturer's instructions. Egg yolk IgY was extracted from a commercial egg by injecting distilled water directly into the peptide‐immobilized affinity column (1 mL), which was connected to a Profinia purification system (Bio‐Rad). Washing buffer (PBS) was used to remove unbound materials. Binding IgY was eluted with 0.1 M glycine‐HCl/0.25 M NaCl (pH 2.5 and pH 3), and the eluate was immediately neutralized with neutralization buffer (1 M Tris–HCl, pH 8.5) and stored at 4 °C until use. The obtained fractions were evaluated for immunoreactivity by enzyme‐linked immunosorbent assay (ELISA) and purity by SDS‐PAGE analysis. From the SDS‐PAGE data, the purity of the obtained fractions was estimated using GelAnalyzer2010a software. Protein concentrations were estimated from the absorbance at 280 nm using an absorbance coefficient of 1.55 mL/mg for IgY and other proteins.

Khan K.H., Himeno A., Kosugi S., Nakashima Y., Rafique A., Imamura A., Hatanaka T., Kato D, & Ito Y. (2017). IgY‐binding peptide screened from a random peptide library as a ligand for IgY purification. Journal of Peptide Science, 23(10), 790-797.

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Biotinylation and Immobilization of Peptides for Binding Assays

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Y4‐4 and Y5‐55 peptides were biotinylated via PEG₄ linker to the N‐terminus, and then 385 and 324 nmol/column were immobilized, respectively, on a HiTrap™ Streptavidin HP column (1 mL; GE Healthcare) according to the manufacturer's instructions. IgY, IgY‐Fc, and human IgG were used as standard proteins to identify binding against peptides. After injecting the standard proteins, the column was washed with PBS and peptide‐absorbed proteins were eluted with elution buffer (0.1 M glycine‐HCl, pH 2.5 and pH 3).

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Purification of Biotinylated OmCI Protein

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Using an Akta pure (GE Healthcare), biotinylated E141A/H164A OmCI was diluted to 20 μm, and 2.0 ml was injected onto a 1-ml Hi-Trap streptavidin HP column (GE Healthcare) at a flow rate of 0.5 ml/min.
The column was equilibrated with 5× column volume of PBS. Ammonium sulfate precipitated serum was loaded at a flow rate of 0.5 ml/min, and a 5× column volume wash with PBS at a flow rate of 1 ml/min was performed. C5 was eluted and collected in fractions, using an isocratic elution of 5× column volume of 20 mm Tris, 2 m MgCl₂, at a flow rate of 1 ml/min.
Immediately after elution, the fractions were pooled and injected onto a HiLoad 16/600 Superdex 200-pg gel-filtration column (GE Healthcare) that had been pre-equilibrated with PBS. The column was run at 1 ml/min, and all peaks were collected. The peaks were analyzed by SDS–PAGE, and those with a molecular weight consistent with C5 monomer were pooled and stored at −80 °C.

Macpherson A., Liu X., Dedi N., Kennedy J., Carrington B., Durrant O., Heywood S., van den Elsen J, & Lawson A.D. (2018). The rational design of affinity-attenuated OmCI for the purification of complement C5. The Journal of Biological Chemistry, 293(36), 14112-14121.

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