AtHB13-GST fusion protein was purified from Escherichia coli expression strain BL21 Star (DE3) pRARE, which was generated by transforming the pRARE plasmid isolated from Rosetta (DE3) pRARE cells (Merck) into E. coli BL21 Star (DE3) (Invitrogen). Recombinant GST-fusion protein was purified using GST-agarose beads following the manufacturer’s instructions (Sigma–Aldrich, Taufkirchen, Germany). Electrophoretic mobility shift assays (EMSA) was performed as described (Wu et al., 2012 (link)) using the Odyssey Infrared EMSA kit (LI-COR). Sequences of 5′-DY682-labeled fragments are given in Supplementary Table S1 .
Gst agarose beads
Manufactured by Merck Group
Sourced in Germany
GST-agarose beads are a type of affinity chromatography resin used for the purification of proteins fused with the Glutathione S-Transferase (GST) tag. The beads are composed of agarose particles with covalently linked glutathione, which binds to the GST tag on the target protein, allowing its capture and isolation from complex mixtures.
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Lab products found in correlation
Sonicator 3000, by Bioventus (2 mentions)
Odyssey infrared emsa kit, by LI COR (1 mentions)
Bl21 star de3, by Merck Group (1 mentions)
Bl21 star de3, by Thermo Fisher Scientific (1 mentions)
Ni nta, by Qiagen (1 mentions)
Ubiquitin aldehyde, by R&D Systems (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
E2 ubch5a, by R&D Systems (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
4 protocols using gst agarose beads
1
Purification and EMSA of AtHB13-GST
2
Protein Purification from BL21 Cells
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These experiment were performed as previously described37 (link), with the following modifications. The constructs were transformed into a BL21 strain and grown at 37 °C until at an optical density at 600 nm of 0.6. They were then induced with 1 mM isopropyl-β-thiogalactopyranoside overnight at 26 °C. Subsequently, cells were pelleted and resuspended with PBS following sonication with a Misonix sonicator 3000. Then, Triton X-100 (1%) was added and cells were incubated on ice for 1 h. The lysates were clarified by centrifugation and then immobilized on GST-agarose beads (Sigma-Aldrich) or Nickel-nitrilotriacetic agarose (Ni-NTA, Qiagen) for 6 h at 4 °C. Finally, the beads were washed and then eluted.
3
Production and Purification of Recombinant GST-Pin1 Proteins
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As previously described [23 (link), 24 (link)], GST fusion construct pGEX-2T-Pin1, its point mutation, or its truncated mutants were transformed into Escherichia coli strain BL21 and grown at 37°C until an optical density at 600 nm of 0.6 was reached. Then, the bacteria were induced with 0.5 mM isopropyl-β-thiogalactopyranoside (IPTG) at 37°C for additional 3–4 hr; subsequently, cells were pelleted and resuspended with ice-cold PBS following sonication using a Misonix sonicator 3000. Then, a final addition of 1% triton X-100 was performed to the homogenate and incubated on ice for 1 h. The lysates were clarified by centrifugation and immobilized on GST-agarose beads (Sigma-Aldrich, St Louis, MO) at 4°C overnight. Finally, the beads with recombinant GST fusion proteins were washed with PBS.
4
In Vitro Ubiquitination Assay
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The GST-CRY1-WT and GST-CDT1 substrates were captured and eluted off of GST-agarose beads (Sigma) after IPTG induction in transformed BL21 cells for 3 hr at 37°C. The FLAG-tagged DDB1 and CUL4A E3 complexes were immunoprecipitated from U2OS cells by anti-FLAG M2 antibody (Sigma) after Ad-Ddb1/Cul4A transduction. Ad-GFP transduced cells were used as IP control. Protein-A sepharose beads were equilibrated in ligase assay buffer (25 mM Tris-HCl pH = 7.5, 50 mM NaCl, 1 mM EDTA, 0.01% NP-40, 10% glycerol) twice and mixed with the GST substrate in 30 μL of the ubiquitin mix containing: 25 mM Tris-HCl, pH = 7.4, 5 mM MgCl2, 2 mM ATP, 2 mM sodium fluoride, 1mM DTT, 10 nM okadaic acid, 250 μM ubiquitin aldehyde (Boston Biochem), 120 ng E1 (His6-UBE1, Boston Biochem), 300 ng E2 (UbcH5a, Boston Biochem), and 10 μg ubiquitin (Boston Biochem) [22 (link)]. The reactions were kept in a 37°C shaker for 2 hr and then denatured by adding 5X SDS loading buffer and boiling at 95°C for 5 mins. The final reactions were run in an 8% SDS-PAGE gel and subjected to immunoblotting with anti-ubiquitin antibody.
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