Streptavidin-conjugated paramagnetic beads (Z5481, Magnesphere, Promega) were washed three times in PBS according to the manufacturer's instructions, and then mixed with 450 μg of A375 EV proteins, in 500 μl (final volume) of binding buffer (BB: 75 mM Tris-HCl, pH 7.5; 50 mM KCl; 5 mM dithiothreitol) (27 (link)), containing proteases and phosphatases inhibitors (Sigma-Aldrich). Samples were incubated for 1 h, at 4°C, under shaking, to allow unspecific protein binding to the particles (pre-clearing step). After centrifuging at 10,000 × g for 5 min, the pre-cleared supernatants were used for the specific binding reaction. The pre-cleared sample was divided into two aliquots, one of which was mixed with H1.0 RNA (1.2 μg) in BB, while the other one was used as an RNA-free control. Both samples were incubated for 1 h at 4°C, after which fresh aliquots of pre-washed beads were added, and incubation was continued for 1 h, at 4°C, under shaking. Finally, the supernatants containing unbound proteins were collected by a magnetic device (Magnesphere, Promega) and frozen. Paramagnetic beads were washed four times in BB and then resuspended in electrophoresis sample buffer, boiled and centrifuged at 10,000 × g. The supernatants, which contain bound proteins, were frozen and saved for analyses.
Magnesphere
Manufactured by Promega
Sourced in France
The MagneSphere is a magnetic separation system designed for efficient purification and isolation of biomolecules. It utilizes magnetic beads to capture and separate target analytes from complex samples, enabling effective sample preparation for downstream applications.
Automatically generated - may contain errors
7 protocols using magnesphere
1
Magnetic Bead Capture of Protein-RNA Complexes
2
UHRF1 Histone H3 Binding Assay
3
Biotin-Labeled Capture Probes for Selective Borrelia Binding
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A set of three capture probes labeled with biotin at the 5′ terminus synthesized by Operon Technologies/Qiagen, Alameda, CA, were used for hybrid selection. These were Probe A: 5’-Biotin- GCC -TTA –ATA-GCA -TGT -AAG –CAA- AAT- GTT- AGC- AGC-CTT GAT -3′; Probe B: 5’-Biotin-TCC-ATC-GCT-TTT-AAT-TCC-TGT-GTA-TTC-AAG-TCT-GGT-TCC-3′, and Probe C: 5’-Biotin- ATC TGT AAT TGC AGA AAC ACC TTT TGA AT -3′. Probe A and Probe B were designed to selectively bind OspA and Probe C to flagellin gene sequences respectively that are conserved in different BB strains.
Fifty μl of a 1 μg/ml solution of each of the capture probes were added to each of the processed sample in a tube and mixed. The tubes were incubated at 85 °C for 10 min to denature the DNA, followed by incubation at 37 °C for 3 h for hybridization of the biotin labeled probes to BB DNA. The hybrids were captured on the Magnesphere® paramagnetic particles derivatized with streptavidin (Promega, Madison, WI). The target DNA-probe hybrid bound to the beads was washed three times with wash buffer (0.1xSSC, 0.1% Sarkosyl, 0.1% BSA, pH 6.8) for 5 min each at 37 °C to remove excess probe, PCR inhibitors and cell debris. The target DNA-probe hybrid was then dissociated from the beads by adding 100 μl of 10 mM Tris buffer and incubating at 65 °C for 10 min. The beads were then separated from the DNA by centrifugation.
Fifty μl of a 1 μg/ml solution of each of the capture probes were added to each of the processed sample in a tube and mixed. The tubes were incubated at 85 °C for 10 min to denature the DNA, followed by incubation at 37 °C for 3 h for hybridization of the biotin labeled probes to BB DNA. The hybrids were captured on the Magnesphere® paramagnetic particles derivatized with streptavidin (Promega, Madison, WI). The target DNA-probe hybrid bound to the beads was washed three times with wash buffer (0.1xSSC, 0.1% Sarkosyl, 0.1% BSA, pH 6.8) for 5 min each at 37 °C to remove excess probe, PCR inhibitors and cell debris. The target DNA-probe hybrid was then dissociated from the beads by adding 100 μl of 10 mM Tris buffer and incubating at 65 °C for 10 min. The beads were then separated from the DNA by centrifugation.
4
Quantification of Histone Modifications
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A total of 40 μl of streptavidin paramagnetic beads (MagneSphere, Promega) was washed three times with PD300 buffer [20 mM Hepes-NaOH (pH 7.9), 300 mM KCl, 0.2% (v/v) Tween 20, and 20% (v/v) glycerol]. A total of 2 μg biotin-labeled mononucleosomes or water was added for overnight incubation at 4°C with rotation, followed by three washes with PD300. Recombinant proteins were incubated in the absence and presence of 10-fold molar excess of di-C 16:0 PI5P or di-C 16:0 PI4P overnight at 4°C. A total of 10 μg recombinant proteins (without and with PIPs) in 500 μl of binding buffer was added to washed beads, and reactions were incubated for 3 hours at 4°C with rotation. Beads were washed six times with PD300, and the recovered material was eluted in 30 μl of PDelute buffer [50 mM tris-HCl (pH 8), 25% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 1 mM EDTA, 2% (w/v) SDS, and 1 mM TCEP] by boiling for 10 min. The intensity of the hUHRF1 band was integrated for each reaction using ImageJ (http://imagej.nih.gov/ij/ ). Values were corrected for mock and normalized to the intensity of the H2A/H2B band. Fold change was determined as ratio of H3K9me3/H3unmodified normalized signal. Experiments were performed with three technical replicates using two independently prepared samples.
5
CHIP Pulldown Assay in Jurkat Cells
6
Quantification of Telomeric DNA in Cancer Cells
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Cells were grown in RPMI medium for 72 h (12 × 106 cells for A2780 and 17 × 106 cells for A2780cis cells) and treated by 50 µM of Pt-ttpy, Pt-tpy for 8 h. The protocol was adapted from [53 (link)]. Cells were recovered and DNA was extracted as above. The purified genomic DNA was subsequently digested overnight at 37 °C by HinfI and RsaI restriction enzymes. Each sample containing the digested DNA was incubated for hybridization with 150 pmole of biotinylated (CCCTTA)4 probe at 70 °C in 0.5× Saline Sodium Chloride-Sodium Citrate buffer (SSC) then allowed to cool to room temperature. Then, the streptavidin beads (Magnesphere, Promega, Charbonnières-les-Bains, France) in Denhardt’s solution were added to the samples and incubated on a rotating wheel at 4 °C with the annealed samples. The supernatant S1 containing genomic fragments and telomeric DNA not yet hybridized were recovered. Telomeric DNA was eluted twice with 150 μL water at 70 °C. The S1 fraction was re-hybridized with the probe attached to the beads, and the same steps as above were repeated. The DNA concentration was determined by measuring the absorbance at 260 nm using the Nano-Drop (1000 V) (Thermofisher, Villebon sur Yvette, France).
7
Probing UHRF1 Linker Peptide Interactions
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A total of 40 μl of streptavidin paramagnetic beads (MagneSphere, Promega) was washed three times with phosphate-buffered saline (PBS) in low-binding tubes. A total of 10 μg of biotin-labeled hUHRF1 linker 2 peptides or water was added for 1 hour at room temperature with rotation, followed by three washes with PBS. A total of 10 μg of recombinant hUHRF1 linker 4 peptide in 500 μl of binding buffer [20 mM Hepes-NaOH (pH 7.9), 150 mM NaCl, 0.05% (v/v) Tween 20] was added, and reactions were incubated for 1 hour at room temperature with rotation in the absence and presence of 10-fold molar excess of di-C 16:0 PI5P or di-C 16:0 PI3P (final concentrations, 2.15 μM linker 4 and 21.5 μM PIP). Beads were washed three times with binding buffer, and the recovered material was eluted in 30 μl of PDelute buffer [50 mM tris-HCl (pH 8.0), 25% (v/v) glycerol, 0.25% (w/v) bromophenol blue, 1 mM EDTA, 2% (w/v) SDS, 1 mM TCEP (Tris(2-carboxyethyl)phosphine)] by boiling for 5 min.
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