DOCK11-BioFLAGHis purified as described previously was immobilized on streptavidin-conjugated magnetic beads (Promega) in TBST. Lysate containing T7-Ack1-His was mixed with DOCK11-immobilized beads on a rotator for 3 h at 4 °C in the presence of DCS8-42A. The beads were washed with TBST and resuspended in NuPAGE LDS Sample Buffer (4×) containing 0.1 M DTT. The eluate was separated in a 3 to 8% SDS-PAGE and analyzed by Western blotting with HRP-conjugated mouse anti-FLAG-tagged monoclonal antibody or anti-Ack1 antibody.
Streptavidin conjugated magnetic beads
Manufactured by Promega
Streptavidin-conjugated magnetic beads are a type of lab equipment used in various biotechnological applications. They consist of magnetic particles coated with the protein streptavidin, which has a high affinity for the molecule biotin. These beads can be used to capture and isolate biotinylated molecules, proteins, or cells from complex biological samples.
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Lab products found in correlation
4 protocols using streptavidin conjugated magnetic beads
1
Dock11-Ack1 Protein Interaction Assay
2
Affinity Purification of DnaA Protein Complexes
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This assay was performed as previously described (Ishida et al., 2004 (link)). Purification of bio-DnaA, bio-DnaA domain I–II, and bio-DnaA domain III–IV has also been reported (Ishida et al., 2004 (link)). Aliquots of protein extracts described above were incubated for 5 min on ice in buffer C (20 μL), in the presence or absence of bio-DnaA, or its truncated derivatives (10 pmol). The mixtures were further incubated for 1 h at 4°C, with gentle rotation, in the presence of streptavidin-conjugated magnetic beads (Promega). The beads and the bound materials were collected using magnetic force and washed twice with buffer C (20 μL) without bovine serum albumin. DnaA-bound proteins were eluted in a standard SDS sample buffer (10 μL) and analyzed using SDS-13% PAGE and silver staining.
3
Peptide Affinity Purification of Protein Complexes
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All peptides were synthesized by Eurofins genomics. HEK293T cells were transfected with HisBioFLAG-DENND2A or HisT7-SASH1 for 24 hours. These cells were washed with PBS and lysed with RIPA buffer. Lysates were centrifuged at 4°C for 30 min at 16,000 × g, and the supernatant was collected. HisBioFLAG-DENND2A in the lysates was immobilized on streptavidin-conjugated magnetic beads (Promega) in TBST. Lysate containing HisT7-SASH1 was mixed with DENND2A-immobilized beads on a rotator for 5 hours at 4°C in the presence of each peptide. The beads were washed with TBST and resuspended in NuPAGE LDS Sample Buffer (1.6×) containing 0.1 M DTT to elute binding proteins.
4
Histone Modification Analysis by Western Blot
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Western blot analyses were carried out as previously described (13) . Briefly, cells were lysed with 1% Triton X-100 or NP-40 with 1X protease inhibitor cocktail (MCE, HY-K0010). Lysates were resolved on SDS-PAGE followed by immunoblotting. Anti-Histone H3 (Cell Signaling Technology, cat. #4499), Anti-Histone H3K27me3 (Cell Signaling Technology, cat. #9733), and other primary antibodies were used. When indicated, cell were treated with an EZH2 inhibitor GSK-126 (MCE, HY-13470) or agonist GSK-J4 (MCE, HY-15648B) for 7 to 14 days. For immunoprecipitation experiments, whole-cell lysates were precleared with protein A/G beads (MCE, HY-K0202) and incubated with IgG control or anti-AcK antibody (1:100, Cell Signaling Technology, 9441) for overnight rocking at 4 C. Protein G beads were added and mixed for another 4 hours. For anti-avi immunoprecipitation experiments, lysates from cells coexpressing Escherichia coli biotin holoenzyme synthetase (BirA) and indicated proteins were incubated with streptavidin-conjugated magnetic beads (Promega, Z5482) for 4-hour rocking at 4 C. Immunocomplexes were resolved on SDS-PAGE, followed by immunoblotting.
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