The separated proteins were transferred from the gel to PVDF membrane and then blocked using PBST (PBS with 0.1% Tween 20) containing 5% non-fat milk for 30 min at room temperature with 60 r.p.m. shaking, anti-GST (1:5000; #M20007; Abmart), anti-his (1:5000; #M30111; Abmart), Anti-Flag (1:5000; #F3165; Sigma-Aldrich), anti-GFP (1:5000; #M20004; Abmart), anti-RFP (1:5000; #5f8; Chromotek), antibodies were added to PBSTM (PBS with 0.1% Tween 20 and 5% non-fat dry milk) and incubated at room temperature for 3–4 h, followed by three times washes with PBST. The membrane was then incubated with a goat anti-mouse IRDye 800CW antibody (Odyssey, no. 926-32210; Li-Cor) at a ratio of 1:10,000 in PBSTM at room temperature for 30 min with 60 r.p.m. shaking. The membrane was washed three times with PBST, and then visualized by excitation at 800 nm. Full-size images are presented in Supplementary Fig. 21 .
Anti gst
Manufactured by Abmart
Sourced in China
Anti-GST is a type of lab equipment used for the detection and purification of proteins tagged with Glutathione S-Transferase (GST). It functions by binding to and capturing GST-tagged proteins, allowing for their isolation and purification from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti his, by Abmart (7 mentions)
Anti gfp, by Abmart (4 mentions)
Anti flag, by Merck Group (3 mentions)
Anti rfp, by Proteintech (2 mentions)
Anti mbp, by Abmart (2 mentions)
Anti flag, by Abmart (2 mentions)
Anti his, by Beyotime (2 mentions)
Anti mbp, by New England Biolabs (2 mentions)
Ni nta agarose, by Qiagen (2 mentions)
Glutathione superflow resin, by Takara Bio (2 mentions)
Gst resin, by GE Healthcare (1 mentions)
Anti his antibody, by Abmart (1 mentions)
Matchmaker gold yeast two hybrid system, by Takara Bio (1 mentions)
Lsm 980, by Zeiss (1 mentions)
Gst tag resin, by Beyotime (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti ubiquitin, by Abcam (1 mentions)
Double color infrared laser imaging system, by LI COR (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
18 protocols using anti gst
1
Western Blot Protein Detection Protocol
2
Recombinant Protein Purification and Interaction
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The primers used to construct His-KIX8/9 were listed in S1 Table . The coding sequences of KIX8 and KIX9 were cloned into pET-28a(+). GST-SAP and GST-GUS were described before[38 (link)]. Pull-down assay was performed as described previously[27 (link)], and the precipitates were analyzed by immunoblot with anti-GST (Abmart) and anti-His (Abmart) antibodies.
3
Production and Purification of Recombinant Fusion Proteins
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To produce the GmLHP1-His fusion protein, the CDS of GmLHP1 was cloned into the pET29b (+) expression vector. The recombinant fusion plasmids were transformed into E. coli Rosetta (DE3) cells. The fusion proteins was purified at 4 °C and quantified according to the pET System Manual. To produce the GmBTB/POZ-GST protein, the CDS of GmBTB/POZ was inserted into the pGEX-4T-1 expression vector and expressed in Rosetta (DE3) cells. The target protein was purified with GST resin (GE Healthcare; 17-0756-01). Pull-down was performed as described by Yang et al.80 (link). The pulled-down proteins were eluted by boiling, separated by 12% SDS-PAGE, and detected by immunoblotting using anti-GST (Abmart, code number M20007S) and anti-His antibodies (Abmart, code number M20001S), respectively.
4
Protein-Protein Interaction Assays
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Vectors for all the following analyses were constructed by introducing the full-length coding sequences of the genes into empty vectors using the in-fusion strategy. Also, see our previous studies for the available vectors related to GSK2 and aGSK2 (Liu et al., 2021 (link); Tong et al., 2012 (link)). All primers and resulting vectors are listed in Supplemental Table 1 . A yeast two-hybrid assay was performed using the Matchmaker Gold Yeast Two-Hybrid System (Clontech) according to the product instructions. The pull-down assay and expression and purification of fusion proteins were performed as described previously (Liu et al., 2022 (link)). BiFC, luciferase complementation, and Co-IP assays were performed as detailed previously (Chen et al., 2008 (link); Xiao et al., 2020 (link); Liu et al., 2022 (link)). The fluorescence was detected with a confocal fluorescence microscope (LSM 980, ZEISS). The luminance signal was detected with an imaging system equipped with a cold charge-coupled device camera (LB 985, NightSHADE with indiGO software). Detection of proteins by immunoblotting was performed using tag antibodies including anti-FLAG (1:1000, Sigma), anti-GFP (1:1000, Abmart), anti-GST (1:2000, Abmart), and anti-MBP (1:2000, Abmart).
5
Purification of GST-ATG6 and HIS-MAPK20 Proteins
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The CDS of ATG6 and MAPK20 was amplified and inserted into pGEX4T-1 or pET32a to generate GST-ATG6 or HIS-MAPK20 recombinant protein, respectively. For pull-down assays, GST-ATG6 protein was first incubated with GST-tag resin (Beyotime, Shanghai, China, P2251) at 4°C for 2 h with slow rotation. Then, HIS-MAPK20 was added and incubated for 2 h. The beads were centrifuged at 4°C,1000 g for 2 min, washed five times with pull-down buffer, and boiled with 2 × SDS loading buffer. The proteins were analysed with an anti-GST (Abmart, Shanghai, China, M20007) or anti-HIS (Abmart, Shanghai, China, M30111) antibody.
6
GST-PWWP3 Protein Interaction Assay
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In total, 500 μg GST-PWWP3 and 500 μg MBP or MBP-DIP1 were mixed into 1 ml of TGH buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA pH 7.5, 1% Triton, 5% glycerol, 1 mM PMSF and protease inhibitor cocktail (Roche)) and incubated at 4 °C for 1 h. After incubation, reaction mixtures were pulled down with 120 μl of Glutathione Sepharose 4B (GE) for 1 h, then washed five times with the TGH buffer. The pulled-down proteins were analyzed on 10% SDS-PAGE gels, and then subjected to immunoblotting with anti-MBP (Abmart) or anti-GST (Abmart) antibodies.
7
In vitro Ubiquitination Assay Protocol
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Ubiquitination assay in vitro was carried out following the protocol described by Zhao et al. (29 ). Wheat E1 protein, AtUBC8, and the predicted soybean E2 Glyma06g13020 were expressed in E. coli BL21 and purified by Ni NTA beads. GmPUB13 or GmPUB13(252-630) and other mutants fused with GST-tag were expressed in E. coli BL21 and purified by immunoprecipitation with glutathione Sepharose 4B beads (Cat. No. 45-000-139, GE Healthcare). Each reaction with 30 µL final volume contained 50 ng wheat E1 protein, 200 to 500 ng AtUBC8 or the predicted soybean E2 Glyma06g13020 protein as E2 conjugating enzyme, 5 μg ubiquitin together with reaction buffer (50 mM Tris⋅HCl pH 7.4, 10 mM MgCl2, 5 mM dithiothreitol, 5 mM ATP, 10% glycerol) in the tubes containing beads binding with GmPUB13, mutants, or GST-tag proteins. The reactions were incubated in a thermomixer for 3 h at 30 °C with shaking and stopped by adding 1× SDS-PAGE loading buffer and incubated for another 5 min at 100 °C. Avr1d and Avr1dF90A aliquoting (5 μL) for each reaction were analyzed by electrophoresis on 12% SDS-PAGE gels. The incubated mixtures were detected by Western blot using anti-ubiquitin (Abcam), anti-GST (Abmart), and anti-Flag (Abmart).
8
Western Blot Analysis of Recombinant Proteins
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Proteins were separated in SDS-PAGE gels and the separated proteins were transferred to PVDF membranes. The membranes were then blocked using 5% non-fat milk in PBST buffer (1 × PBS + 0.1% Tween 20) (PBSTM) for 30 min at room temperature with 60 r.p.m. shaking. The appropriate antibodies were then added to the PBSTM. Antibodies used were: anti-GST (1:5000; #M20007; Abmart), anti-His (1:5000; #M30111; Abmart), Anti-Flag (1:5000; #F3165; Sigma-Aldrich), anti-GFP (1:5000; #M20004; Abmart), and anti-RFP (1:5000; #5f8; Chromotek). After addition of the antibodies, the membranes were incubated at room temperature for 2–4 hr or 4°C for overnight; then washed three times (5 min each) with PBST buffer. The membranes were then incubated with a goat anti-mouse (Odyssey, no. 926 – 32210; Li-Cor) or anti-rabbit (Odyssey, no. 926–32211; Li-Cor) IRDye 800CW antibody (Odyssey, no. 926 – 32210; Li-Cor) at a dilution of 1:10,000 in PBSTM at room temperature for 30 min with 60 r.p.m. shaking; and followed by three washes (5 min each) with PBST. The signals were detected by excitation at 700 and 800 nm using a double color infrared laser imaging system (Odyssey, LI-COR company).
9
Protein-Protein Interaction Assay
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Pull‐down assay was prepared as previously described (He et al., 2021 (link)). The recombinant GST fusion proteins were added to the Glutathione Mag‐Beads (Sangon Biotech, China) for 2 h at 4 °C with agitation and then washed with binding/washing buffer three times. The recombinant His‐OsUBP1a/b proteins were then added to the beads complex and incubated for another 2 h at 4 °C. After five times of beads washing, the complexes were eluted with an elution buffer. Proteins were detected by western blot with appropriate antibodies, anti‐GST (Abmart, MA9024), anti‐His (Beyotime, AF2870).
10
In Vitro Ubiquitination Assay Protocol
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In vitro ubiquitination was carried out as described (Gimenez-Ibanez et al., 2009 (link); Stegmann et al., 2012 (link)). In brief, each reaction of 60 μl of final volume contained ubiquitination buffer (50 mM of Tris–HCl [pH 7.5], 5 mM of MgCl2, 2 mM of DTT, 4 mM of ATP, and 1 × protease inhibitor cocktail) and 1 μg of MBP-EXO70B1 or MBP-EXO70B2 in the presence or absence of 15 μg of ubiquitin, 50 ng of human E1 (UBE1), 200 ng of Arabidopsis E2 His-UBC8, and 500 ng of GST-AvrPtoB or GST-AvrPtoBF479A. The reactions were incubated at 30°C for 3 h and stopped by adding 5 × SDS sample buffer and boiling for 10 min. Samples were separated by an 8% SDS-PAGE gel followed by detection of ubiquitinated substrate by immunoblotting using anti-MBP (New England Biolabs) and anti-GST (Abmart) antibodies.
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In vitro ubiquitination was carried out as described (Gimenez-Ibanez et al., 2009 (link); Stegmann et al., 2012 (link)). In brief, each reaction of 60 μl of final volume contained ubiquitination buffer (50 mM of Tris–HCl [pH 7.5], 5 mM of MgCl2, 2 mM of DTT, 4 mM of ATP, and 1 × protease inhibitor cocktail) and 1 μg of MBP-EXO70B1 or MBP-EXO70B2 in the presence or absence of 15 μg of ubiquitin, 50 ng of human E1 (UBE1), 200 ng of Arabidopsis E2 His-UBC8, and 500 ng of GST-AvrPtoB or GST-AvrPtoBF479A. The reactions were incubated at 30°C for 3 h and stopped by adding 5 × SDS sample buffer and boiling for 10 min. Samples were separated by an 8% SDS-PAGE gel followed by detection of ubiquitinated substrate by immunoblotting using anti-MBP (New England Biolabs) and anti-GST (Abmart) antibodies.
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