To assess the direct interaction of PASK with the subunits of human H3K4 MLL2 complex, 0.3 picomoles of hPASK-V5 was incubated with 0.3 picomoles of recombinant MLL2, ASH2, WDR5 or RBBP5 in high salt IP buffer (1×) in the presence of 100 mM NaCl, detergent, DTT and Protease inhibitors (Nuclear Complex Co-IP Kit, Active motif Cat #, 54001) for 4 h at room temperature. The reaction products were then incubated with 1 μg of V5 peptide antibody (SC- 271944, Santa Cruz) overnight at 4°C. The complex was captured with V5 agarose beads (A7345, Sigma), washed five times with IP wash buffer (Nuclear Complex Co-IP Kit, Active motif Cat #, 54001) and eluted with SDS loading dye and analyzed by western blotting. Ten percent of the reaction was saved as input before the bead capture.
V5 agarose beads
Manufactured by Merck Group
V5-agarose beads are a type of affinity chromatography resin designed for the purification of recombinant proteins tagged with the V5 epitope. The beads are composed of cross-linked agarose and have the V5 antibody covalently bound to their surface, which allows for the specific capture and isolation of V5-tagged proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Rfp trap beads, by Proteintech (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Bioruptor next gen, by Diagenode (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Hsp70, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Kinase assay buffer, by Cell Signaling Technology (1 mentions)
Adenosine triphosphate (atp), by Cell Signaling Technology (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Ubch5b, by R&D Systems (1 mentions)
Anti ubiquitin antibody, by Cell Signaling Technology (1 mentions)
E1 ube1, by R&D Systems (1 mentions)
Lambda phosphatase, by New England Biolabs (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
8 protocols using v5 agarose beads
1
Investigating PASK-MLL2 Complex Interactions
2
PIWI Protein Immunoprecipitation Protocol
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Lysates from Aag2 cells expressing V5–3xFlag tagged PIWI proteins were pre-cleared with protein G agarose beads and then incubated with V5-agarose beads (Sigma). The immunoprecipitates were washed, and RNA was isolated from the beads for subsequent analyses. For a detailed description of the experimental procedure, see Supplementary data.
3
Affinity Purification of Tagged Proteins
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GFP- and RFP-tagged transgenes were immunoprecipitated using GFP- and RFP-TRAP beads (Chromotek), respectively, according to the manufacturer’s instructions. V5-tagged transgenes were purified using V5-agarose beads (Sigma). For Ago3 and Piwi5 IP experiments, antibodies targeting endogenous proteins were added to lysates at 1:10 dilution and incubated for 4 h at 4°C, followed by overnight binding to Protein A/G PLUS agarose beads (Santa Cruz). Protein extracts were resolved on polyacrylamide gels, blotted to nitrocellulose membranes, and probed with the indicated antibodies. Details on generation of antibodies, experimental procedures, and antibody dilutions can be found in the Supplementary Data.
4
SETBP1 Interactome Identification
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One hundred million HEK 293T cells were transiently transfected with pcDNA 6.2 V5-SETBP1-WT, SETBP1 G870S, or with the empty vector as control. Forty eight hours after transfection, cells were collected, washed twice with cold PBS, lysed in Buffer 1 (Pipes, pH 8.0, 5 mM, KCl 85 mM, NP-40 0.5%), supplemented with protease inhibitors (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific), kept for 10 min on ice, homogenized with douncer homogenizer (10 hits), and centrifuged at 700 × g for 10 min. The pellet was then resuspended in Buffer 2 (Tris HCl, pH 8.0, 50 mM, sodium dodecyl sulfate 0.1%, deoxicholate 0.5%) supplemented with protease inhibitors, sonicated with Bioruptor Next Gen (Diagenode) (5 cycles 30 s ON, +30 s OFF) to promote gDNA disruption and centrifuged at 18,000 × g for 10 min. The supernatant representing the nuclear fraction was quantified with Bradford assay and a total of 1 µg of protein was loaded on 100 µl V5-agarose beads (Sigma-Aldrich) and incubated under rotation overnight at 4 °C. Beads were then washed three times with PBS+protease inhibitors, and elution was performed with 7 M Urea, 2 M Thiourea, and 4% CHAPS for subsequent mass spectrometric analysis or with Laemmli buffer for western blot analysis. All chemicals were purchased from Sigma Aldrich.
5
Immunoprecipitation of SAMD9 and SAMD9L Proteins
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For immunoprecipitation of hSAMD9 or hSAMD9L, HeLa cells or IFN-treated ΔhSAMD9 HeLa cells were infected with different VACV viruses. For immunoprecipitation of mSAMD9L, 293FT cells were transfected with the mSAMD9L expression plasmid and then infected with different VACVs at 48 hr post transfection. After 8 hr of infection, the cells were lysed on ice with a lysis buffer (0.1% (w/v) NP-40, 50 mM Tris, pH 7.4, 150 mM NaCl) supplemented with protease inhibitor cocktail tablets (Roche Molecular Biochemicals). The cleared cell lysates were mixed with 50 μl of 50% (vol/vol) V5-agarose beads (Sigma-Aldrich) for 30 min at 4°C. After washing with lysis buffer, the beads were resuspended in SDS sample buffer, the eluted proteins were resolved by SDS-PAGE and detected with Western blot as described previously [34 (link)]. The detection antibodies were mouse monoclonal antibodies (mAb) against V5 (Sigma-Aldrich; clone V5-10), Flag tag (Sigma-Aldrich) or HSP70 (Santa Cruz), and rabbit polyclonal antibodies against hSAMD9 (Sigma-Aldrich, HPA-21319) and hSAMD9L (Proteintech, 25173-1-AP).
6
AKT2 Variant Kinase Assay
7
Ubiquitination of Substrate rhN3
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The reactions were carried out at 37°C for 45 min in 25 µl of ubiquitination reaction buffer (40 mM Tris-HCl at pH 7.6, 2 mM DTT, 5 mM MgCl2, 0.1 M NaCl, 2 mM ATP) containing the following components: 250 µM ubiquitin, 100 nM E1 (UBE1), 200 nM UbcH5b (all from Boston Biochem). Purified FLAG-WWP2 was added to the reaction. rhN3 (0.5 µg) was used as a substrate in the reactions as indicated. After the ubiquitination reaction, lysis buffer was added, and samples were incubated with V5-agarose beads (Sigma) for 2 hr. The beads were washed five times with lysis buffer and TBS and boiled in SDS–PAGE loading buffer. ubiquitination of rhN3 was monitored by Western blotting with anti-ubiquitin antibody (Cell Signaling). An aliquot of each reaction was used directly for Western blotting to demonstrate equal amounts of substrate and/or E3 ligase in the samples as indicated.
8
Cdk8 Nuclear Depletion Protocol
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Overnight cultures were diluted to OD600 0.15 and grown to an OD600 of 1.5. For Cdk8 nuclear depletion rapamycin was added to a final concentration of 7.5 µM when cell reached OD600 0.6. Cells were lysed in Lysis buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton, 0.1% sodium deoxycholate, 0.1% SDS) + Complete protease inhibitor mixture (Roche Applied Science) and 0.1 mM PMSF. Fusion proteins were captured using V5 agarose beads (Sigma # A7345) that were pre-cleared overnight with PBS + 0.1% BSA. Samples were washed twice with Lysis buffer without phosphatase inhibitors and once with 1X NEBuffer for protein metallophosphatases (50 mM HEPES, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, 1 mM MnCl2).
Beads were resuspended in 1X NEBuffer for protein metallophosphatases and separated into the indicated treatment groups. 400 units of lambda phosphatase (New England Biolabs, #P0753) was added as indicated, with all samples incubated 15 min at 30°C. Samples were resuspended in Lysis buffer plus 2X SDS buffer and analyzed by SDS-PAGE.
Beads were resuspended in 1X NEBuffer for protein metallophosphatases and separated into the indicated treatment groups. 400 units of lambda phosphatase (New England Biolabs, #P0753) was added as indicated, with all samples incubated 15 min at 30°C. Samples were resuspended in Lysis buffer plus 2X SDS buffer and analyzed by SDS-PAGE.
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