DNA-protein pull-down was performed as previously described (43 (link)). In brief, 293T cells were transfected with HA-Zscan4c overexpression vector. Two days after transfection, 293T cells were lysed by sonication in lysis buffer containing 50 mM Tris–HCl pH8.0, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.5% NP-40 and protease inhibitors for the preparation of nuclear exact. Nuclear extracts were precleared with Streptavidin MagBeads (GenScript, L00424) for 1 h. After that, the precleared supernatant was incubated with 1 μg biotinylated double-stranded oligonucleotide and poly(dI-dC) on a rotating shaker at 4°C for 4 h. DNA-bound proteins were collected by incubating with Streptavidin MagBeads at 4°C for 1 h. Then the beads were washed five times with PBS, and the bound proteins were released from beads by boiling the beads in SDS loading buffer. Western blot was performed to detect the proteins with specific antibodies. The sequences of biotinylated double-strand oligonucleotides used in the pull-down assay are listed in Supplementary Table S1 .
Streptavidin magbeads
Manufactured by GenScript
Sourced in China
Streptavidin MagBeads are magnetic beads coated with the protein streptavidin. Streptavidin has a high affinity for the small molecule biotin, allowing the beads to be used for the capture and separation of biotin-labeled molecules.
Automatically generated - may contain errors
Lab products found in correlation
Cellytictm m, by Merck Group (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Protein a g sepharose beads, by Merck Group (1 mentions)
Igf2bp3, by Proteintech (1 mentions)
Lipofectamine 3000 kit, by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Proteintech (1 mentions)
8 protocols using streptavidin magbeads
1
DNA-Protein Pull-Down Assay
2
C99 Biotinylation and Affinity Purification
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PS gene deleted HTL cells were seeded at a density of 0.8 × 106/well in 6-well plates and transfected the following day with 300 ng of BirA biotin ligase encoding DNA, 500 ng of Avi–C99 encoding DNA, and 500 ng of C99–TEV site–rTA–FLAG encoding DNA with Lipofectamine® 2000 (Invitrogen) transfection reagent. After transfection, biotin solution was added to 40 μm . Cells were harvested and lysed in CelLyticTM M (Sigma-Aldrich) 1 day after transfection. The crude supernatant protein extracts were incubated with prewashed Streptavidin MagBeads (GenScript) for about 30 min, followed by three washes with lysate buffer. All samples with beads were subjected to SDS-PAGE for Western blot analysis using anti-FLAG and anti-β-actin antibodies. The β-actin level was used as loading control.
3
Biotin-Labeled I3MO Pulldown Assay
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I3MO was labeled with D-biotin (#60338ES03, YEASEN).35 (link) D-biotin worked as negative control. Cell lysates from MM cell lines (ARP1 and RPMI8226), and the recombinant proteins for PSME3 (P03968, Solarbio) or PSME4 (P05708, Solarbio) were incubated with I3MO-D-biotin (200 µM) at 4°C overnight, respectively. Then, streptavidin MagBeads (#L00424, GenScript)36 (link) were added to cell lysates and rocked gently at room temperature for extra 3 h, followed by PBS washing three times (1 mL for each time) to thoroughly remove the non-specifically bound proteins. The beads left were re-suspended in SDS-PAGE sample buffer (90 µL), and heated at 95 °C for 5 min. The pull-down protein was identified by western blots with primary antibody of PSME3 and PSME4, respectively.
4
Antibody-based assays for protein detection
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The monoclonal or polyclonal antibodies against FNDC3B (catalog No. 22605‐1‐AP), IGF2BP3 (catalog No. 14642‐1‐AP), CD44 (catalog No. 15675‐1‐AP), and flag–tag (catalog No. 20543‐1‐AP) were purchased from Proteintech Group (Wuhan, China). Horseradish peroxidase (HRP)‐conjugated goat antimouse IgG and HRP‐conjugated goat antirabbit IgG were obtained from Pierce (Thermo Fisher Scientific, MA). RNA RT and polymerase chain reaction (PCR) kits were purchased from TaKaRa (Dalian, China). ChamQ SYBR qPCR Master Mix (Real‐time PCR kits) was obtained from Vazyme (Nanjing, China). Gibson Assembly DNA (HB‐infusion TM) kits were obtained from HANBIO (Shanghai, China). Lipofectamine‐3000 kits were obtained from Thermo Fisher Scientific. Protein A/G‐Sepharose beads were obtained from Millipore (Merck, NJ). Streptavidin MagBeads was obtained from GenScript (NJ).
5
Biotin-Adaptor Binding for Sequencing
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Biotin was covalently bound to the 3-end of the adaptor sequence to enable adaptor binding to streptavidin. The reverse complementary (RV) sequences of the adaptor were synthesized at the 5-end of primers (RV-primer); thus, the adaptor was annealed to the RV-primer due to base pairing, and the unpaired part of the RV-primer could anneal to the template.
Streptavidin MagBeads (GenScript, Nanjing, China, Cat. No. L00424) was supplied as a 25% slurry in phosphate-buffered saline (PBS) at pH 7.4. For each reaction, 10 μl bead slurry was used. The beads were washed three times with 100 μl buffer containing 10 mM Tris at pH 7.5, and 50 mM NaCl were washed three times to remove residual ethanol. For biotin-containing DNA binding to the beads, incubation was performed at RT for 60 min on a roller. The beads were washed thrice with the same buffer mentioned above to avoid unbound DNAs. Reagents for strand extension were added directly to the beads or to the supernatants after the beads were boiled in a thermal cycler at 94°C for 5 min and pelleted.
Streptavidin MagBeads (GenScript, Nanjing, China, Cat. No. L00424) was supplied as a 25% slurry in phosphate-buffered saline (PBS) at pH 7.4. For each reaction, 10 μl bead slurry was used. The beads were washed three times with 100 μl buffer containing 10 mM Tris at pH 7.5, and 50 mM NaCl were washed three times to remove residual ethanol. For biotin-containing DNA binding to the beads, incubation was performed at RT for 60 min on a roller. The beads were washed thrice with the same buffer mentioned above to avoid unbound DNAs. Reagents for strand extension were added directly to the beads or to the supernatants after the beads were boiled in a thermal cycler at 94°C for 5 min and pelleted.
6
Streptavidin MagBeads Pulldown Assay
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Streptavidin MagBeads (GenScript) were washed 3 × with PBS containing 1% BSA (PBS-B). 10 µL of beads was added to each cytosolic fraction and rotated for 2 h at 4 °C. Then, beads were washed 3 × with PBS-B and either prepared for western blotting or further used for pulldown ELISA.
7
Characterization of HsfA1d Phosphorylation
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A 200-bp DNA sequence containing double-stranded perfect HSEs within the HSP18.2 promoter was amplified using biotin-labeled primer pairs. The PCR products were purified with NaAc and ethyl alcohol. HsfA1d-His, HsfA1dT263A-His, HsfA1dT263D-His, GST, BIN2-GST, and BIN2K69R-GST were expressed in E. coli BL21 and purified as described above. HsfA1d-His (2 μg) and 0.2 μg GST or BIN2-GST or BIN2K69R-GST were used in each kinase reaction, with or without ATP in the kinase reaction buffer. Kinase reactions were performed at 37°C for 2 h. At the same time, 2 μg biotin-labeled double-stranded PCR products were added to 20 μL streptavidin MagBeads (catalog #L00424; GenScript) in 100 μL TES binding buffer (0.01 M Tris, 1 mM EDTA, 2 M NaCl), bound at room temperature for 1 h, and washed 3 times with IP buffer (0.1 M L-glutamic acid monopotassium salt monohydrate, 0.05 M Tris [pH 7.5], 2 mM MgCl2, 0.05% [w/v] NP-40). Some kinase reaction products (10 μL) were removed as input, and the rest of the reaction products were added to DNA-bead mixes in 500 μL IP buffer for binding at 4°C for 2 h. Finally, 5× SDS loading buffer was added to the bead mixes after washing 3 times with IP buffer, and the bead mixes were then denatured by boiling and separated by SDS-PAGE for detection with anti-His and anti-GST antibodies.
8
DNA Bridging Assay for Protein-DNA Interactions
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For the DNA bridging assay, two DNA duplexes, Site-1 (forward sequence: 5′-GGGAAAGTTTCTATTATTAG CAGAGATA-3′) and Site-2 (5′-CTAAGCAAATGAGATGAATATGCAGGGCACCATGCTAAAAATAA AATGGTTTCATGGTGCTAGTGAGGAAGGAA-3′), were synthesized. The 5′ end of the forward chain of Site-1 DNA was synthesized with a biotin label. The pull-down experiment was conducted as follows: 10 pmol Site-1 DNA was bound to 10 μl Streptavidin MagBeads (GenScript, Cat. No. L00424) according to the reaction conditions following the manufacturer's recommendation; the beads were blocked with 5% BSA and 1 μM T7 primers; the beads were washed thrice with binding buffer (5 mM Tris pH 7.4, 0.5 mM EDTA, 250 mM NaCl); immobilized Site-1 DNA was mixed with recombinant proteins (HDAC4GRD and MEF2A1–95) and 2 pmol Site-2 DNA, and further incubated at 4°C for 1 h; the beads were washed 8 times, and Site-2 DNA was released by boiling in pure water containing 0.1% SDS. The enrichment of Site-2 was detected by qPCR using the SYBR green system with the following primers: 5′-CTAAGCAAATGAGATGAATATGCA-3′ and 5′-TTCCTTCCTCACTAGCACCATG-3′.
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