For the in vitro pull-down assays, genes were cloned in pGEX-4T-2 (GST tag) and pET32a (His tag) constructs, respectively. Proteins were expressed in E. coli strain BL21 and extracted as previously described. Proteins with GST tag were incubated with glutathione agarose beads (Thermo Fisher Scientific, 16101) at 4 °C for 4 h and washed by wash buffer (50 mM NaCl, 50 mM Tris⋅HCl pH = 7.5, 0.05% Tritonx100) three times. Then, proteins with His tag were incubated with the beads at 4 °C for 4 h. Finally, the beads were washed three times and detected by anti-GST (1:2,000, Abmart, M20007) and anti-His antibodies (1:2,000, Abmart, M20001) by Western blot. For the in vivo coimmunoprecipitation assays, proteins were expressed in N. benthamiana and extracted by the PH-increased lysis buffer (150 mM NaCl, 10 mM Tris⋅HCl pH = 8.3, 0.5 mM ethylenediaminetetraacetic acid [EDTA], 1% Tritonx100) with 1% protease inhibitor mixture (Sigma-Aldrich, P9599). The total proteins were incubated with GFP-Trap_A beads (Chromotek, ACT-CM-GFA0050) at 4 °C for 2 h and washed by washing buffer (150 mM NaCl, 10 mM Tris⋅HCl pH = 7.5, 0.5 mM EDTA, 1% protease inhibitor mixture) three times. Proteins were detected by anti-GFP (1:2,000, Abmart, P30010) and anti-RFP (1:2,000, Chromotek, 6G6) antibodies. All of the experiments were repeated three times with similar results.
M20007
Manufactured by Abmart
Sourced in China
The M20007 is a laboratory equipment item. It is designed to perform specific functions in a laboratory setting. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
M20001, by Abmart (7 mentions)
M30111, by Abmart (2 mentions)
Glutathione agarose, by Merck Group (1 mentions)
Gst antibody, by Abmart (1 mentions)
Gst bind resin, by Merck Group (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Ni nta resin, by Merck Group (1 mentions)
A21010, by Abbkine (1 mentions)
Glutathione sepharose beads, by Sangon (1 mentions)
Ni nta agarose, by Qiagen (1 mentions)
M20002, by Abmart (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Gst tag purification resin, by Beyotime (1 mentions)
Gst tag protein purification kit, by Beyotime (1 mentions)
Hrp conjugated anti mouse igg, by Merck Group (1 mentions)
Glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Ni nta agarose, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 1 vector, by Cytiva (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (1 mentions)
M20008, by Abmart (1 mentions)
16 protocols using m20007
1
In Vitro and In Vivo Protein-Protein Interactions
2
Protein Interaction Assay with TMK4
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TAA1-His and His-Sumo proteins were expressed in E. coli and captured by Ni-NTA resin. TMK4 KD (587aa–928aa) sequence was amplified and inserted into pGEX4T-2 vector. GST-TMK4 KD proteins were purified from E. coli by Glutathione agarose (sigma G4510) and incubated with TAA1-His or His-Sumo beads in binding buffer (20 mM HEPES, pH 7.5, 40 mM KCl, 1 mM EDTA, 1% glycerol, 0.1‰ TritonX-100 with PMSF) for 2 h at 4 °C. The beads were washed three times by washing buffer (20 mM HEPES, pH 7.5, 40 mM KCl, 1 mM EDTA, and 0.05‰ TritonX-100). GST-TMK4 KD proteins were detected by GST antibody (M20007, Abmart, 1:2000 dilution) via western blots, and His-TAA1 and His-Sumo proteins were detected by Coomassie blue staining.
3
Affinity Purification of Fusion Proteins
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The full-length coding sequences of AtHSPR, KNAT5, and OFP1 were fused with either an N-terminal glutathione S-transferase (GST) tag using a pGEX-6P-1 vector or a C-terminal His tag using a pET-30a vector to express GST-AtHSPR, GST-KNAT5, His-KANT5, and His-OFP1. The fused constructs were transformed into the Escherichia coli Rosetta strain for protein expression. The GST-tagged recombinant proteins were purified with glutathione (GSH) magnetic beads (70601, Beaver-Bio) according to the manufacturer’s instructions. The primers used for plasmid construction are shown in Supplementary Table S1 .
For GST pull-down assays, the GSH magnetic beads bound with GST or GST-proteins were rinsed five times with washing buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 2 mM dithiothreitol, 1.5% Triton X-100, and 1 mM PMSF) and then the His-proteins were incubated with pre-washed GST-bound or GST-protein-bound GSH magnetic beads in lysis buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, and 1 mM PMSF) for 2 h at 4 °C with gentle shaking. After incubation, the magnetic beads were rinsed five times with the washing buffer. The magnetic beads were then boiled with 2×SDS loading buffer for 10 min, and then the pulled-down proteins were subjected to immunoblotting analysis with anti-His and anti-GST antibodies (Abmart, M20001 and M20007, respectively).
For GST pull-down assays, the GSH magnetic beads bound with GST or GST-proteins were rinsed five times with washing buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, 2 mM dithiothreitol, 1.5% Triton X-100, and 1 mM PMSF) and then the His-proteins were incubated with pre-washed GST-bound or GST-protein-bound GSH magnetic beads in lysis buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4, and 1 mM PMSF) for 2 h at 4 °C with gentle shaking. After incubation, the magnetic beads were rinsed five times with the washing buffer. The magnetic beads were then boiled with 2×SDS loading buffer for 10 min, and then the pulled-down proteins were subjected to immunoblotting analysis with anti-His and anti-GST antibodies (Abmart, M20001 and M20007, respectively).
4
Characterizing Protein-Protein Interactions
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For each combination, 2 µg of GST, HIS, or MBP-tagged proteins were mixed in 400 µL binding buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 25 mM imidazole, 0.5% NP-40) on a rotating wheel at 4 °C for 4 h, and then incubated with Ni-NTA HIS•Bind Resin at 4 °C for 2 h, followed by western blot analysis with anti-GST (Abmart, M20007, 1:2000 dilution), -HIS (Abmart, M30111, 1:2000 dilution), and -MBP (Proteintech, 15089-1-AP, 1:2000 dilution) antibodies, respectively.
5
Protein Interaction Assay via Pull-Down
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N1 and N1-P fragments were constructed using the pET28a-SUMO vector (Novagen) to fuse with a His-SUMO tag. SUVH2 was cloned into pGEX4T-1 (GE Healthcare) to fuse with a glutathione S-transferase (GST) tag. All of the constructs were transformed into Escherichia coli Rosseta (DE3) and proteins were expressed using isopropyl β-d -1-thiogalactopyranoside induction at 18 °C for 16 h. GST- and His-SUMO proteins were purified using GST-Bind Resin (Millipore, 70541) and Ni-NTA Resin (Millipore, 70666). Approximately 5 μg of each protein were mixed together in pull-down buffer (50 mM Tris⋅HCl pH 7.5, 2 mM EDTA, 200 mM NaCl, 0.2% Nonidet P-40) at 4 °C for 3 h. His binding resin at 15 μL was added and incubated with rotation for 1.5 h at 4 °C. Protein-bound beads were washed with pull-down buffer four times and incubated with boiling sodium dodecyl sulfate (SDS) loading buffer (EpiZyme, LT101) for 10 min. Eluted proteins were detected using western blots with anti-GST (Abmart, M20007) and anti-His (Abmart, M20001). The primers are listed in SI Appendix, Table S1 .
6
GST-OsWRKY7 Protein Degradation Assay
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GST‐OsWRKY7 recombinant protein was induced in E. coli BL21(DE3) and purified by glutathione affinity resin column (Pierce，16100). The cell‐free degradation assays were performed as previously described (Wang et al., 2009 (link)). Briefly, total proteins from 100 mg WT leaves were extracted in 1 mL degradation buffer. Then, the supernatants were collected by centrifugation at 13 000 rpm for 10 min at 4 °C. A total quantity of 100 ng purified GST and GST‐OsWRKY7 were incubated in 100 μL protein extracts without (−) or with (+) 100 μM MG132 at 28 °C for 0, 0.5, 1, 2, and 3 h. The protein abundance was detected by an anti‐GST antibody (1:10 000, Abmart, M20007), followed by a secondary antibody (1:5000, Abbkine, A21010). Coomassie blue‐stained Rubisco large protein (RubL) was used as a loading control.
7
Recombinant Protein Expression and Interaction Analysis
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The coding sequence of ARFTF17 was amplified using primers ARFTF17-PET30F and ARFTF17-PET30R and cloned into the pET30a vector to create a fusion protein with a His tag. The coding sequence of MYB40 was amplified using primers MYB40-pCOLDF and MYB40-pCOLDR, and cloned into the pCold vector to create a fusion protein with the GST tag at NH-terminus. Recombinant proteins were produced in Escherichia coli BL21 (DE3) (EC1002) induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 20 °C for 20 h. The proteins were purified on Ni-NTA agarose (QIAGEN, 30210), following the manufacturer’s instructions. For pull-down assays, equal amounts of bait and prey proteins were incubated at 4 °C overnight in binding buffer (20 mM Tris-HCl, pH 8.0, 150 mM of NaCl, 0.2% [v/v] Triton X-100, 10% (v/v) glycerol, EDTA free protease inhibitor1025 cocktail (Roche, 4693132001). The protein complexes were recovered with glutathione Sepharose beads (Sangon Biotech, C650031), which were washed three times with buffer (50 mM Tris-HCl, pH 8.0, 140 mM of NaCl, 0.1% [v/v] Triton X-100, and EDTA free protease inhibitor1025 cocktail (Roche, 4693132001). After removing unbound proteins, those bound were analyzed by immunobloting using anti-GST (Abmart, M20007) and anti-ARFTF17 antibodies. The primers used in this assay are listed in Supplementary Table 1 .
8
Protein-protein interaction assay
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For the pull‐down assay, the full‐length CDS of RiSte11 was fused to pETMALc‐H, RiPbs2 was fused to pET‐32a and pGEX, RiHog1 was fused to pGEX to generate MBP‐RiSte11, His‐RiPbs2, GST‐RiPbs2 and GST‐RiHog1 constructs. Recombinant proteins were separately expressed in Escherichia coli DE3 cells (Transgen, China) by growing cultures to OD600 = 0.6 at 20°C. And recombinant proteins were induced with 0.2 mM IPTG for 12 h. Purified MBP‐RiSte11, MBP (negative control) proteins were incubated with MBP amylose beads (New England Biolabs, E8035) and GST‐RiHog1, GST (negative control) proteins were incubated with GST amylose agarose beads (Thermo Scientific, 78 601) at 4°C for 1 h. After washing the beads five times with PBS buffer, GST‐RiPbs2 with MBP‐RiSte11 or MBP, His‐RiPbs2 with GST‐RiHog1 or GST were pulled down at 4°C for 2 h. Then the bound proteins were boiled in 5× SDS sample buffer. Samples were subjected to the subsequent separation on an SDS–PAGE gel and immunoblotting analysis using anti‐MBP (Abmart, M20051), anti‐His (Abmart, M20001) and anti‐GST (Abmart, M20007) antibodies as described in Situ et al. (2020 (link)).
9
Protein-Protein Interaction Assay
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The purified Flag-tagged protein was mixed with the purified GST-tagged protein (or Myc-tagged APRF1) in 1 ml of pull-down buffer [20 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1 mM DTT and EDTA-free protease inhibitor cocktail (Roche)]. A 100-μl volume of the mixture was used as input, and the remaining mixture was incubated with 40 μl of Glutathione Sepharose® 4B at 4°C with gentle rotation for 1 h. After the Glutathione Sepharose® 4B was washed six times with 1 ml of pull-down buffer at 4°C, proteins bound on the beads were eluted with 200 μl of elution buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 20 mM glutathione and 1 mM DTT] at 4°C for 30 min. Finally, the eluted and input samples were boiled with SDS loading buffer and separated on SDS-PAGE gels for western blotting with Flag antibody (Sigma, F7425) and GST antibody (Abmart, M20007) or Myc antibody (Abmart, M20002).
10
GST-Fusion Protein Interaction Assay
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GST, GST-MoMkk1, and His-MoAtg4 were expressed in Escherichia coli BL21 cells. Cells were collected and washed 3 times with 1× PBS, then resuspended and lysed with a sonicator. After centrifugation with 13,000 g for 10 min, the GST or GST-MoMkk1 supernatant was mixed with 30 μl of glutathionesepharose beads (GE Healthcare, 10265165), which were washed 3 times using GST washing buffer (250 mM NaCl, 20 mM Tris, 2 mM EDTA, 2 mM EGTA, pH 7.5), then incubated at 4°C for 2 h. The supernatant was discarded after centrifugation (13,000 g, 10 min) and the GST- or GST-MoMkk1-bound to GST beads were incubated with His-MoAtg4 protein solution for another 4 h. The beads were washed 5 times with GST washing buffer. Proteins were eluted with GST elution buffer (10 mM reduced glutathione in GST washing buffer, pH 8.0), then analyzed by Western blot analysis with anti-His (Abmart, M20001) and anti-GST (Abmart, M20007) antibodies, respectively.
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