The CDS of ABF1 and SAPK10 were fused into pGEX-4T-1 (G.E. Healthcare, Chicago, IL, USA) and pET-28a (Thermo, Waltham, MA, USA) vectors, respectively. The GST-ABF1, GST and His-SAPK10 were transformed into Transetta (DE3) chemically competent cells (Transgen, Beijing, China), and purified using the glutathione S-transferase (GST)-SefinoseTM Kit (Sangon Biotech, Shanghai, China) and 6× His-Tagged Protein Purification Kit (CWBIO, Beijing, China), respectively. The purified proteins were incubated with 50 µL Glutathione High Capacity Magnetic Agarose Beads (Sigma-Aldrich, St. Louis, MO, USA) and 600 µL pull-down buffer (50 mM Tris-HCl, pH 7.5, 5% glycerol, 1 mM EDTA, 1 mM Dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 0.01% NonidetP-40, and 150 mM KCl) at 4 °C for 2 h. Then the beads were washed five times with pull-down buffer, and suspended in 50 μL of 1× PBS and 10 μL of 6× SDS protein loading buffer, boiled for 5 min, and resolved on 10% acrylamide gels. Individual bands were detected using Supersignal West Pico Chemiluminescent Substrate (Thermo, Waltham, MA, USA) and the ChemDocTM Touch Imaging system (Bio-Rad, Hercules, CA, USA). The dilution for anti-GST (Yeasen, Shanghai, China) and anti-His (Yeasen, Shanghai, China) was 1: 5000. The primers used were listed in Table S1 .
Anti gst
Manufactured by Yeasen
Sourced in China
Anti-GST is a laboratory reagent used to detect and quantify the presence of Glutathione S-Transferase (GST) in biological samples. It provides a reliable and efficient method for researchers to study enzyme activities, protein interactions, and cellular processes involving GST.
Automatically generated - may contain errors
Lab products found in correlation
Anti lamp2, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Immunoway (2 mentions)
Anti β actin, by Yeasen (2 mentions)
Anti vegf, by Novus Biologicals (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
Anti ha 11, by BioLegend (2 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (2 mentions)
Anti cd31, by Abcam (2 mentions)
Glutathione high capacity magnetic agarose beads, by Merck Group (1 mentions)
Pet28a, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Transetta de3 chemically competent cell, by Transgene (1 mentions)
Gst sefinose kit, by Sangon (1 mentions)
His tagged protein purification kit, by CWBIO (1 mentions)
Chemdoc touch imaging system, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Yeasen (1 mentions)
Anti hif 1β, by Cell Signaling Technology (1 mentions)
Anti hoip, by Abcam (1 mentions)
Anti sharpin, by Proteintech (1 mentions)
6 protocols using anti gst
1
Purification and Pull-Down of Recombinant Proteins
2
Antibody Validation for Western Blotting
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anti-FLAG M2 (Sigma, F1804), anti-HA.11 (BioLegend, 901501), anti-myc (Santa Cruz Biotechnology, sc-40), anti-GST (Yeasen, 30902ES60), anti-α-tubulin (Yeasen, 30304ES60), anti-β-actin (Yeasen, 30101ES60), anti-GAPDH (Immunoway, YM3029), anti-CD31 (Abcam, ab28364), anti-VEGF (Novus Biologicals, NB100-664), anti-PCNA (Servicebrio, GB11010), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-GFP (Yeasen, 31002ES60), anti-LUB9 (Lifesensors, AB130), anti-HIF1α (Novus, NB100-479; Abcam, ab228649), anti-HIF1β (Cell Signaling technology, #5537), anti-Otulin (Abcam, ab211328), anti-HOIP (Abcam, ab46322; R&D systems, MAB8039), anti-HOIL-1L (Millipore, MABC576), anti-Sharpin (Proteintech, 14626-1-AP), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822), anti-Lamin B1 (Zenbio, R24825), normal mouse immunoglobulin (IgG) 1 (Santa Cruz Biotechnology, sc-3877), anti-mouse IgG(H + L) (Jackson ImmunoResearch, 151383), and anti-rabbit IgG(H + L) (Jackson ImmunoResearch, 145472).
3
Arabidopsis Protein Extraction and Immunodetection
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Total protein was extracted from Arabidopsis seedlings with protein extraction buffer [150 mM NaCl, 0.5% Triton X-100, 50 mM tris-HCl (pH 7.5), and protease inhibitor cocktail]. Anti-Pol II Ser2P (Medical & Biological Laboratories Co., Ltd., MABI0602), anti-FLAG (Sigma-Aldrich, F1804), anti-Myc (EASYBIO, BE2010), anti-GST (Yeasen, 30903ES50), and anti-His (Yeasen, 30403ES40) were used in the assays.
4
Degradation Kinetics of Transcription Factor bZIP72
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The experiments were conducted as described previously (Lv et al., 2014 ). Briefly, the wild‐type (japonica cv Nipponbare) were hydroponically cultured under mock or +ABA (2 µM ABA) nutrition solutions for 2 wk. Then the seedlings were harvested and ground into fine powders in liquid nitrogen, then resuspended in the degradation buffer (25 mM Tris‐HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM DTT and 10 mM ATP). After brief centrifugation twice (12 000 g for 10 min each time), the resulting total proteins (30 µg of each) were incubated with recombinant GST‐bZIP72 or GST‐bZIP72S71D (100 ng of each) at 28°C for the individual assays. The reactions were taken at indicated intervals for determination of bZIP72 abundance by immunoblotting. The protein intensities were quantified using imagej software. The dissociation‐one phase exponential decay curve was plotted on a semilog graph using graphpad prism (5.0) software to calculate the half‐life of GST‐bZIP72. The dilution for anti‐GST (Yeasen) was 1 : 5000.
5
Purification and Interaction of GST-ACE1 and His-TrSAM
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The recombinant GST and GST-ACE1317-463aa were purified using Glutathione Beads (Smart-LifeSciences, China) from E. coli BL21 (DE3) following an 18-hour induction with 0.1 mM IPTG. Similarly, the recombinant His-TrSAM protein was purified using High Affinity Ni-NTA Resin (GenScript, Nanjing, China) from E.coli under identical condition. A weight of 20 μg purified GST or GST- ACE1317-463aa fusion proteins was incubated with 20 μg His-TrSAM in 500 μL incubation buffer (50 mM Tris-HCl pH6.8, 250 mM NaCl, 1.5% glycerol, 0.6% Triton X-100 and 0.1% Tween-80) for 4 h at 4 °C. The beads were then washed three times with the incubation buffer. The washed beads were boiled in SDS loading buffer and subsequently separated followed by western blot analysis with anti-GST (Yeasen, Shanghai, China, #30901ES) and anti His-tag mouse monoclonal antibody (Yeasen, Shanghai, China, #30401ES).
6
Comprehensive Antibody Panel for Research
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anti-FLAG M2 (Sigma, F1804), anti-HA.11 (BioLegend, 901501), anti-Myc (clone 9E10, Santa Cruz Biotechnology, sc-40), anti-GST (Yeasen, 30902ES60), anti-NRP1 (Cell Signaling Technology, #3725), anti-NRP2 (Cell Signaling Technology, #3366), anti-EGLN3 (Novus, NB100–303), anti-Erk3 (Santa Cruz Biotechnology, sc-365234), anti-arginase1 (Cell Signaling Technology, #9819), anti-α-tubulin (Sigma, T6074), anti-β-actin (Yeasen, 30101ES60), anti-GAPDH (Immunoway, YM3029), anti-CD31 (Abcam, ab28364), anti-Mac3 (BD Phmarmingen, 550292), anti-CD8 (InVitrogen, #14–0081–82), anti-F4/80 (Abcam, ab6640), anti-CD25 (Servicebrio, GB11612), anti-VEGF (Novus Biologicals, NB100–664), anti-PCNA (Servicebrio, GB11010), anti-cleaved caspase-3 (Cell Signaling Technology, #9661), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-MMP2 (Abcam, ab37150), anti-phospho-IкBα (Cell Signaling Technology, #2859), anti-iNOS (Cell Signaling Technology, #2982), anti-p53 (Santa Cruz Biotechnology, sc-126), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822), anti-p16 (Santa Cruz Biotechnology, sc-1661), anti-p21 (Santa Cruz Biotechnology, sc-817), normal mouse IgG1 (Santa Cruz Biotechnology, sc-3877), and anti-mouse IgG(H + L) (Jackson ImmunoResearch, 715-585-151).
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