The gene sequences of ureB, hspA, and ureB-hspA were amplified by PCR from the vaccine SH02, cloned into an expression vector pGEX-6P-1(+) plasmid (GE Healthcare, Pittsburgh, USA), and placed between BamHI and NotI restriction sites (UreB-F: 5′-CGCGGATCCAAAAAGATTAGCAGAAAAG-3′, UreB-R: 5′-AAATATGCGGCCGCCTAGAAAATGCTAAAGAG-3′; HspA-F: 5′-CGCGGATCCAAGTTTCAACCATTAG-3′, HspA-R: 5′-AAATATGCGGCCGCTTAGTGTTTTTTGTGATC-3′; UreB-HspA-F: 5′- CGCGGATCCAAAAAGATTAGCAGAAAAG-3′, UreB-HspA-R: 5′- AAATATGCGGCCGCTTAGTGTTTTTTGTGATC-3′). The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells (Cwbio, Suzhou, China), and protein expression was subsequently induced with 1 mM IPTG at 20°C before being harvested after 18 hours by centrifugation. The bacterial pellets were resuspended in PBS and sonicated. Any insoluble cellular components were removed by centrifugation, and the rGST-UreB, rGST-HspA, and rGST-UreB-HspA fusion proteins were purified by Price Glutathione Superflow Agarose (Thermo, Rockford, USA) according to the manufacturer’s protocol. Moreover, the GST tag was excised with PreScission Protease (Beyotime, Nanjing, China) and removed with GST-tag Purification resin (Beyotime, Nanjing, China). Finally, the purity and concentration of the recombinant protein rUreB, rHspA, and rUreB-HspA were determined via SDS-PAGE, Western Blot, and BCA assay.
Gst tag purification resin
Manufactured by Beyotime
Sourced in China
The GST-tag purification resin is a laboratory product designed for the purification of proteins tagged with glutathione S-transferase (GST). It is used to isolate and concentrate GST-tagged proteins from complex biological samples, such as cell lysates or culture supernatants. The resin consists of agarose beads covalently bound with glutathione, which selectively binds to the GST tag on the target protein, allowing for its separation and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions)
Histrap hp, by GE Healthcare (2 mentions)
Pgex 6p 1 plasmid, by GE Healthcare (1 mentions)
Prescission protease, by Beyotime (1 mentions)
Peroxidase pod assay kit, by Solarbio (1 mentions)
Bl21 competent cells, by Vazyme (1 mentions)
Ni nta agarose beads, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Emsa kit, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 (cck8), by Selleck Chemicals (1 mentions)
Clean blot ip detection reagent hrp, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mg132, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Genmute sirna transfection reagent, by SignaGen (1 mentions)
Glutathione (gsh), by Beyotime (1 mentions)
Anti gst, by EASYBIO (1 mentions)
19 protocols using gst tag purification resin
1
Cloning and Purification of Recombinant Helicobacter Proteins
2
Degradation Assays for GhDFR and VdFAE
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GhDFR‐His and GST‐VdFAE fusion proteins were produced in E. coli BL21 (DE3) cells by being induced with 0.1 M isopropyl β‐d ‐thiogalactopyranoside (IPTG). The GST‐VdFAE and GhDFR‐His fusion proteins were purified with GST‐tag purification resin (Beyotime) and HisPur Ni‐NTA agarose (Qiagen), respectively. The cells were then harvested by centrifuging at 10,500 g for 10 min at 4°C and subjected to sonication. For the in vitro degradation assay, GhDFR‐His with GST or GST‐VdFAE were co‐incubated for 0, 1, 2 and 4 h at room temperature. The reaction system contained 2 μg purified GhDFR‐His and 0.5 μg purified GST‐VdFAE in a volume of 40 μL. The reaction products were analysed by immunoblotting using the anti‐His or anti‐GST antibodies (Proteintech). For the in vivo degradation assay, A. tumefaciens cells carrying GhDFR‐FLAG were first infiltrated into N. benthamiana leaves. One day later, A. tumefaciens cells carrying VdFAE‐GFP or GFP were infiltrated into the leaves that consistently expressed GhDFR‐FLAG. Samples were taken at 24 and 48 hpi in order to investigate the amount of GhDFR protein. The reaction products were analysed by immunoblotting using the anti‐GFP or anti‐FLAG antibodies (Proteintech).
3
Cloning and Purification of Sspep1 and Pod-1a
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The CDSs of Sspep1 and pod-1a were amplified by PCR using the corresponding primer pairs pGEX-pep1-BamHI-F/pGEX-pep1-BamHI-R and pET30a-pod-BamHI-F/pET30a-pod-BamHI-R (Table S1 ) with cDNA generated from the whit part of a whip of infected sugarcane plants serving as a template. The fragments were cloned into the prokaryotic expression vectors pGEX4-1 and pET30a to yield the constructs pGEX4-Sspep1 and pET-Pod-1a. The plasmids were transformed into BL21 Competent Cells (Vazyme, Nanjing, China), and the expressed recombinant proteins were purified using Ni–NTA Agarose beads or GST-tag Purification Resin (both from Beyotime, Shanghai, China). In vitro horseradish peroxidase (HRP) activity was assayed by the Peroxidase (POD) Assay Kit (Solarbio, Beijing, China).
4
Recombinant EV71 Capsid Protein Expression
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Full-length VP1, VP2, VP3, and VP4 segments of the EV71 Fuyang strain were codon-optimized and synthesized by Genewiz (Suzhou, China) and cloned into a pGEX4T-1 prokaryotic expression vector. Recombinant pGEX4T-EV71-VP1, pGEX4T-EV71-VP2, pGEX4T-EV71-VP3, and pGEX4T-EV71-VP4 plasmids were transformed into Escherichia coli strain BL21(DE3) (TianGen, Beijing, China). Recombinant EV71 VP1, VP2, VP3, and VP4 proteins were expressed with isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.5 mM when the optical density (OD)600 of bacterial cultures reached 0.6–0.8. Bacterial sediment was collected and sonicated for 30 min after culture for 16 h at 22°C. The supernatant was centrifuged and filtered by 0.45 and 0.22 μm filter membranes to remove the bacterial debris and mixed with glutathione S-transferase (GST)-tag purification resin (Beyotime Biotechnology) for 2 h at room temperature. GST fusion proteins were collected with an elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl, pH8.0) after washing five times with 30 mL phosphate-buffered saline (PBS; pH7.0). Purified GST fusion proteins were detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and their concentrations were determined using a BCA kit (Beyotime Biotechnology), according to the manufacturer’s instructions.
5
Cloning and Purification of NaWRKY70 Transcription Factors
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The full-length coding sequences of NaWRKY70, NaEIN3-like, NaEIN3-like1, NaMYC2a, NaMYC2b, and NaMYC2c were amplified with specific primers (Supplementary Table S1 ), cloned into the glutathione S-transferase (GST)-fusion pGEX-4T-1 vector and transformed into the Escherichia coli strain BL21. The fusion proteins were induced by isopropyl-β-d -thiogalactopyranoside (0.01–0.05 mM), and the bacterial cells were harvested and purified using GST-tag purification resin (Beyotime). Binding of recombinant protein and biotin-labeled probes was detected using a chemiluminescence electrophoretic mobility shift assay (EMSA) kit (Beyotime) according to the protocol suggested by the manufacturer.
6
Affinity Purification of PUB Interactors
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Tissues, including brain, heart, liver, spleen, lungs, kidneys, stomach, colorectum and bones were obtained from 8-week-old wild-type mice and ground to powder in liquid nitrogen, followed by the lysis in HEPES lysis buffer (20 mM HEPES (pH 7.2), 150 mM NaCl, 0.5% Triton X-100, 1 mM NaF and 1 mM dithiothreitol). GST only, GST-tagged HOIP PUB, NZF, UBA, RBR-LDD were purified from bacteria BL21 and bound to GST-tag Purification Resin (Beyotime, P2251), followed by incubation in tissue lysates at 4 °C overnight. After washing four times with HEPES lysis buffer, the resin was boiled for the immunoblot and mass spectrometry.
7
Recombinant ANXA2 Protein Expression
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ANXA2 protein expression plasmid was constructed by inserting the coding sequence of GST‐fused ANXA2 into pGEX‐6P‐1 plasmid by OBiO Technology Corp. Ltd., Shanghai, China. using the BamHI and XhoI enzymic sites. The recombined plasmid, pGEX‐6P‐ANXA2 or pGEX‐6P‐1, was then transfected into BL21 (DE3) E. coli. Isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) was added to the culture medium when OD600 of the bacteria culture reached 0.6 to induce ANXA2 expression. After induction, and the culture flask kept shaking for another 5 h, the bacteria were collected and lysed with lysozyme. Finally, the resulting supernatant was loaded to GST tag purification resin (Beyotime), and the resin was washed with PBST buffer six times before adding elution buffer and collecting elutions. Purity and product size were evaluated by SDS/PAGE and Coomassie blue staining.
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ANXA2 protein expression plasmid was constructed by inserting the coding sequence of GST‐fused ANXA2 into pGEX‐6P‐1 plasmid by OBiO Technology Corp. Ltd., Shanghai, China. using the BamHI and XhoI enzymic sites. The recombined plasmid, pGEX‐6P‐ANXA2 or pGEX‐6P‐1, was then transfected into BL21 (DE3) E. coli. Isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) was added to the culture medium when OD600 of the bacteria culture reached 0.6 to induce ANXA2 expression. After induction, and the culture flask kept shaking for another 5 h, the bacteria were collected and lysed with lysozyme. Finally, the resulting supernatant was loaded to GST tag purification resin (Beyotime), and the resin was washed with PBST buffer six times before adding elution buffer and collecting elutions. Purity and product size were evaluated by SDS/PAGE and Coomassie blue staining.
8
Purification of GST-tagged BnA09MYB47a Proteins
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To produce glutathione S-transferase (GST)-tagged proteins, BnA09MYB47a was amplified and cloned in the pGEX4T-1 vector and expressed in the Escherichia coli strain Rosetta. Recombinant GST-BnA09MYB47a protein was purified by GST-tag Purification Resin (Beyotime, Jiangsu, China). GST fusion proteins was detected by anti-GST antibodies (Abcam, ab9085), which was diluted 1000 folds and used for protein-DNA binding. EMSA was performed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific, Waltham, USA). The recombinant protein was incubated with labeled probes at room temperature for 20 min; the unlabeled probes were used as a competitor to examine the specificity of binding. Protein-probe complexes were separated by electrophoresis on a native 6% acrylamide gel and the DNA was electroblotted onto nitrocellulose membranes and detected by chemiluminescence. Sequences of DNA probes used in EMSA are listed in Supplementary Data 22 .
9
Cell Culture and Protein Purification Protocol
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SK‐Mel‐28, A375 and HEK293 cells were kindly provided from Cell Bank, Chinese Academy of Sciences. All cells were cultured using DMEM (Gibco) with 10% fetal bovine serum (FBS) in a cell incubator under standard conditions at 37°C and 5% CO2. Reagents used are as follows: GST‐tag Purification Resin (Beyotime Biotechnology, Cat. P2250), GSH (Beyotime Biotechnology, Cat. S0073), protease inhibitor cocktail (Bimake, Cat. B14012), LipoMax plasmid transfection reagent (SUDGEN, Cat.17052012), GenMute siRNA transfection reagent (SignaGen Laboratories, Cat. SL100568), MG132 (Sigma, Cat. C2211), M‐PER™ Mammalian Protein Extraction Reagent (Thermo Fisher, Cat.78501), Protein G Magnetic Beads (Thermo Fisher, Cat. 10004D), Clean‐blot™ IP detection reagent (HRP) (Thermo Fisher, Cat. 21230), RIPA Lysis Buffer (Beyotime, Cat.P0013B) and Cell Counting Kit‐8 (CCK‐8) (Bimake, Cat. B34304).
10
Protein Interaction Mapping of GW6a and CLG1
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The CDS of GW6a was individually cloned into the BamHI and SalI sites of pGEX-4T-1 and the BamHI and HindIII sites of pET-32a to generate fusion proteins GST-GW6a and His-GW6a. The mutated GW6a CDS was cloned into the BamHI and SalI sites of the pGEX-4T-1 vector to express the fusion proteins GST-GW6aS142/T186D and GST-GW6aS142/T186A. The 4 × Myc and CDS of CLG1 sequences were inserted into the multiple cloning site of pMAL-c2X to express a fusion CLG1-Myc protein. The CDS of OsMAPK6 was cloned into the BamHI and SalI sites of pGEX-4T-1 to express GST-OsMAPK6. The constructs were transformed into Escherichia coli BL21 (DE3) and the recombinant proteins were induced by 0.5-mM IPTG (isopropyl-β-D-thio-galactopyranoside). For pull-down assays, GST-tag purification resin (Beyotime) were incubated with GST or GST-GW6a/GST-OsMAPK6 at 4°C for 1 h, and then incubated with CLG1-Myc/His-GW6a at 4 °C for another 1 h. After incubation, the GST beads were washed thoroughly with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.05% SDS, 1% Triton X-100), resolved by SDS-PAGE, and detected and probed with anti-GST (EASYBIO), or anti-Myc (TransGen), or anti-GW6a as indicated. Uncropped scans of immunoblotting results are shown in Source data .
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