The plant tissues were harvested, frozen in liquid nitrogen and ground into powder. Proteins were extracted from the powder using lysis buffer (50 mM Tris‐HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP‐40, 1 × protease inhibitor cocktail, 5 µM MG132) and quantified by Bradford reagent. Equal amounts of protein were separated by SDS‐PAGE, and then transferred to nitrocellulose membrane. The membrane was blocked with 5% non‐fat milk in TBST, and then incubated with the appropriate primary antibody for 3 h, followed by incubation with secondary antibody for 1 h. The primary antibodies included anti‐GFP, anti‐His, anti‐GST (TransGen Biotech, Beijing, China), anti‐Flag (Sigma‐Aldrich), and anti‐p44/42‐ERK (Cell Signaling Technology, Danvers, MA, USA). The secondary antibodies were anti‐mouse IgG and anti‐rabbit IgG (Sigma‐Aldrich). The signal was detected using a Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA).
Anti gst
Manufactured by Transgene
Sourced in China
Anti-GST is a laboratory equipment product designed for the detection and purification of proteins fused with Glutathione S-Transferase (GST) tags. It provides a reliable and efficient method for isolating GST-tagged proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Anti gfp, by Transgene (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti p44 42 erk, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Bca protein assay kit, by CWBIO (1 mentions)
Anti ub, by Merck Group (1 mentions)
Anti phospho p44 42, by Cell Signaling Technology (1 mentions)
Anti tubulin, by Abmart (1 mentions)
Anti mbp, by Transgene (1 mentions)
Anti myc, by Transgene (1 mentions)
Anti phospho ser thr, by Abcam (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Anti his antibody, by Transgene (1 mentions)
Gst resin, by Transgene (1 mentions)
Mg132, by Merck Group (1 mentions)
Protease inhibitor cocktail for plant cell and tissue extracts, by Merck Group (1 mentions)
Glutathione sepharose, by GE Healthcare (1 mentions)
4 protocols using anti gst
1
Protein Extraction and Western Blot Analysis
2
Isolation and Immunoblot Analysis of Apple Proteins
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Protein isolation from apple fruit and calli and immunoblot analysis were performed as previously described (Li et al. 2015 (link)). The protein concentration of each extract was measured using a BCA protein assay kit (Cat. no. P0012S, CWBIO). Purified recombinant MdNAC72 was used to generate a specific antibody in rabbit (Abmart, China, http://ab-mart.com.cn/ ). Anti-His (Cat. no. HT501; TransGen Biotech, China), anti-GST (Cat. no. HT601; TransGen Biotech), anti-MBP (Cat. no. HT701; TransGen Biotech, Beijing, China), anti-GFP (Cat. no. HT801; TransGen Biotech, Beijing, China), anti-FLAG (Cat. no. 14793S; Cell Signaling Technology, USA), anti-Myc (Cat. no. HT101; TransGen Biotech), anti-Ub (Cat. no. ST1200, Sigma, USA), anti-phospho-p44/42 (Cat. no. 4730; Cell Signaling Technology), anti-phosphoSer/Thr (Cat. no. ab117253, Abcam, UK), and anti-tubulin (Cat. no. M20045, Abmart) antibodies were diluted 1:1,000 with Tris-buffered saline with Tween 20 (TBST buffer, 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% [v/v] Tween 20) and incubated with nitrocellulose membranes (Cat. no. S80209, Pall Corporation, USA). Secondary antibodies (goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated; Cat. no. HS201 or HS101, TransGen Biotech) were diluted to 1:3,000 in TBST.
3
Protein-Protein Interaction Analysis
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For protein expression and purification, the GST-PvNAP1/2 fusion proteins were generated using the pGEX4T-1vector, and the recombinant 6 × His-PvSSG fusion protein was generated using the pCold-TF vector and expressed in the Escherichia coli strain "Bl21" after isopropyl-β-d-thiogalactopyranoside induction at 16°C. Recombinant proteins were purified using GST Resin and Ni Resin, respectively (Transgene, Beijing, China). For pull-down assay, GST-PvNAP1/2 or GST proteins (100 μg) were incubated with glutathione-Sepharose 4B resin (Transgene) at 4°C for 1 h to make the beads fully linked with the GST-PvNAP1/2 or GST proteins and then washed 3 times with 500-μL Lysing buffer. Then, 6 × His-PvSSG protein at the same volume was incubated with GST-PvNAP1/2 or GST-linked beads for 2 h. The conjugated constructs were centrifuged and washed 3 times with lysing buffer to prepare for the immunoblot analysis. Finally, the protein extracts before and after immunoprecipitation were analyzed using both anti-GST (Transgene) and anti-His antibodies (Transgene).
4
Immunoprecipitation of DREB2A with BPM2
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Three grams of 35S:GFP-DREB2A(WT) or 35S:GFP-DREB2A(K163R) seedlings was treated in liquid MS medium with 100 mM MG132 (Sigma-Aldrich) for 80 min at 22°C or 42°C, respectively. Total proteins were prepared in the extraction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% [v/v] glycerol, and 1% [v/v] Nonidet P-40) containing protease inhibitor cocktail for plant cell and tissue extracts (Sigma-Aldrich, P9599) and 100 mM MG132. After centrifugation at 13,000g for 15 min, the supernatant was collected for immunoprecipitation. The GST-BPM2 plasmid was constructed by inserting the CDS of BPM2 into pGEX4T-1. GST and GST-BPM2 recombinant proteins were separately expressed in BL21(DE3) and purified by glutathione Sepharose (GE Healthcare), then mixed with the prepared supernatant at 4°C for 30 min. After incubation, the Sepharose was collected and rinsed five times with washing buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10% [v/v] glycerol, and 0.2% [v/v] Nonidet P-40). Proteins were eluted with SDS sample buffer for further analysis via immunoblotting using anti-GST (TransGen Biotech) or anti-GFP (TransGen Biotech) antibodies.
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