GST-fused constructs were expressed in BL21 Escherichia coli. In vitro transcription and translation experiments were done with rabbit reticulocyte lysate (TNT systems, Promega) according to the manufacturer’s recommendation. In GST pull-down assays, about 5 μg of the appropriate GST fusion proteins with 30 μl of glutathione-Sepharose beads was incubated with 5–8 μl of in vitro transcribed/translated products in binding buffer (75 mM NaCl, 50 mM HEPES, pH 7.9) at 4 °C for 2 h in the presence of the protease inhibitor mixture. The beads were washed 5 times with binding buffer, resuspended in 30 μl of 2 × SDS-PAGE loading buffer, and detected by western blotting. For Pull-Down assay of biotinylated LPS, the reaction mixtures containing recombinant galectin-3 and biotinylated LPS were incubated for 4 h at 4 °C. Pull-down of LPS was carried out by treating the mixtures with streptavidin beads (Cytiva).
Streptavidin beads
Manufactured by Cytiva
Sourced in China
Streptavidin beads are solid-phase supports coated with the protein streptavidin. Streptavidin has a high affinity for the small molecule biotin, forming a very stable non-covalent bond. These beads can be used to capture and isolate biotinylated molecules, proteins, or cells in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Genepix 4000 scanner, by Molecular Devices (4 mentions)
Versarray compact microarrayer, by Bio-Rad (4 mentions)
Tnt system, by Promega (1 mentions)
Calf thymus total histones, by Worthington (1 mentions)
Glutathione sepharose beads, by Cytiva (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti gst antibody, by Merck Group (1 mentions)
Concanavalin a cona agarose, by Vector Laboratories (1 mentions)
Anti c myc tag affinity gel, by BioLegend (1 mentions)
Glutathione sepharose 4b beads, by GE Healthcare (1 mentions)
17 protocols using streptavidin beads
1
GST Pull-Down Assay for Protein-Protein Interactions
2
Biotin-Based Proteome Profiling of Keloid Fibroblasts
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Keloid fibroblasts overexpressing DJ-1-V5-TurboID were treated with 50 μM biotin for 30 min. The protein was extracted using RIPA lysis buffer and then treated with ultrasound for 3 min. After centrifugation, part of the protein supernatant was denatured for the detection of whole-cell lysis and the other part was incubated with streptavidin beads (Cytiva) at 4°C overnight. Then, the beads were washed using 1 ml of RIPA lysis buffer, 1 ml of KCl, 1 ml of Na2CO3, 1 ml of 2 M urea (pH 8.0) and 1 ml of RIPA lysis buffer in sequence. Then, 100 μl of elution buffer was added to the beads and heated for 10 min at 95°C. The beads were separated magnetically and elution buffer containing biotinylated proteins was collected for further mass spectrometric analysis. In the liquid chromatography-mass spectrometry (LC–MS) experiment, after reduction and alkylation, the samples were treated with trypsin (mass ratio 1 : 50) and digested at 37°C for 20 h. After desalting, the digested product was lyophilized, redissolved in a 0.1% FA solution and stored at −20°C until further use. The mass/charge ratios of the peptides and peptide fragments were obtained as follows: 20 fragment profiles were collected after each full scan. The raw file was searched by Proteome Discoverer1.4 software and the identified protein results were obtained.
3
Histone Peptide Pull-Down Assay
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The peptide pull-down assays were performed in accordance with the protocol described67 (link). In brief, 1.0 µg of biotinylated histone peptides were incubated with 1 μg of protein or 0.5 ml whole cell extract in binding buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF, protease inhibitors) overnight at 4 °C. After 1 h incubation with Streptavidin beads (Amersham), the beads were washed with the binding buffer and boiled for 5 min. The supernatant from boiled beads was subject to immunoblot analysis. The peptides used are the following:
Unmodified H3 (1–23): ARTKQTARKSTGGKAPRKQIASK;
H3K4me3 (1–23): ARTK(me3)QTARKSTGGKAPRKQIASK;
H3pT11 (1–23): ARTKQTARKST(p)GGKAPRKQIASK;
Unmodified H3 (72–85): REIAQDFKTDLRFQ;
H3K79me3 (72–85): REIAQDFK(me3)TDLRFQ.
Unmodified H3 (1–23): ARTKQTARKSTGGKAPRKQIASK;
H3K4me3 (1–23): ARTK(me3)QTARKSTGGKAPRKQIASK;
H3pT11 (1–23): ARTKQTARKST(p)GGKAPRKQIASK;
Unmodified H3 (72–85): REIAQDFKTDLRFQ;
H3K79me3 (72–85): REIAQDFK(me3)TDLRFQ.
4
Histone Peptide Pulldown Assay
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For the peptide pulldown assays, 1 µg of biotinylated histone peptides with different modifications was incubated with 1.5 µg of GST-fused proteins in binding buffer (50 mM Tris-HCl 7.5, 250 mM NaCl, 0.1% NP-40) overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 h with rotation. The beads were then washed three times and analyzed using SDS-PAGE and western blotting using anti-GST (sc-459) at 1/2000 dilution.
5
Histone Peptide Binding Assay
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An aliquot of 1 µg of biotinylated histone peptides with different modifications were incubated with 1–2 µg of GST-fused proteins in binding buffer (50 mM Tris-HCl 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM phenylmethyl sulphonyl fluoride (PMSF)) at 4 °C overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 h with rotation. The beads were then washed three times and analyzed using SDS-PAGE and western blotting. For GST pulldown, 2 µg protein were incubated with 10 µg of calf thymus total histones (Worthington) in binding buffer (50 mM Tris-HCl 7.5, 1 M NaCl, 1% NP-40, 0.5 mM EDTA, 1 mM PMSF plus protease inhibitors (Roche)) at 4 °C overnight, followed by an additional 1 h Glutathione Sepharose beads (Amersham) incubation. The beads were then washed five times and analyzed using SDS-PAGE and western blotting.
6
Peptide Microarray and Pulldown Analysis of Histone Modifications
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Peptide microarray and was performed as described previously45 (link). In brief, biotinylated histone peptides were printed in triplicate onto a streptavidin-coated slide (PolyAn) using a VersArray Compact Microarrayer (Bio-Rad). After a short blocking with biotin (Sigma), the slides were incubated with the glutathione S-transferase (GST)-tagged GAS41 in binding buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF, 20% FBS) overnight at 4 °C with gentle agitation. After being washed with the same buffer, the slides were probed with an anti-GST primary antibody and then a fluorescein-conjugated secondary antibody and visualized using a GenePix 4000 scanner (Molecular Devices).
For peptide pulldowns, 1 µg of biotinylated histone peptides with different modifications were incubated with 1–2 µg of GST-fused WT GAS41 or indicated mutants in binding buffer (50 mM Tris-HCl 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF) overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 h with rotation. The beads were then washed three times and analyzed using SDS-PAGE and Western blotting.
For peptide pulldowns, 1 µg of biotinylated histone peptides with different modifications were incubated with 1–2 µg of GST-fused WT GAS41 or indicated mutants in binding buffer (50 mM Tris-HCl 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF) overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 h with rotation. The beads were then washed three times and analyzed using SDS-PAGE and Western blotting.
7
Histone Peptide Binding Assay
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Peptide pull-down assays were performed as described previously29 (link). In brief, 1 μg of biotinylated histone peptides with different modifications were incubated with 1–2 μg of GST-fused p300 ZZ domain in binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF) for overnight with rotation at 4 °C. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 h with rotation at 4 °C. The beads were then washed three times and the bound proteins were analyzed using SDS–PAGE and Western blotting.
8
Histone Peptide Microarray and Pull-down Assays
9
Histone Peptide Microarray and Pull-down Assays
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Peptide microarray and peptide pull-down assays were performed as described previously25 (link). Briefly, biotinylated histone peptides were printed in triplicate onto a streptavidin-coated slide (PolyAn) using a VersArray Compact Microarrayer (Bio-Rad). After a short blocking with biotin (Sigma), the slides were incubated with the GST-ENL YEATS domain in binding buffer (50 mM Tris-HCl 7.5, 250 mM NaCl, 0.1% NP-40, 1 mM PMSF, 20% fetal bovine serum) overnight at 4°C with gentle agitation. After being washed with the same buffer, the slides were probed with an anti-GST primary antibody and then a fluorescein-conjugated secondary antibody and visualized using a GenePix 4000 scanner (Molecular Devices). For the peptide pull-down assays, 1 μg of biotinylated histone peptides with different modifications were incubated with 1–2 μg of GST-fused proteins in binding buffer (50 mM Tris-HCl 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF) overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 hr with rotation. The beads were then washed three times and analyzed using SDS-PAGE and Western blotting.
10
Characterizing Histone-Protein Interactions
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Histone peptides with different modifications were synthesized by CPC Scientific. For peptide pulldown assays, 1 µg of biotinylated histone peptides with different modifications were incubated with 1 µg of GST-fused proteins in binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF) overnight. Streptavidin beads (Amersham) were added to the mixture, and the mixture was incubated for 1 h with rotation. The beads were then washed three times and analyzed using SDS-PAGE and Western blotting.
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