Full-length CDS of ELF3, XBAT31, E1 (Arabidopsis UBA1), E2 (human UBCH5b), and Ub (UBQ14) were, respectively, cloned into pETMALc-H, pET-SUMO, or pGEX4T-1 vectors to generate GST-ELF3, MBP-XBAT31, MBP-XBAT31M, His-E1, His-E2, or His-E3 constructs that generate fusion proteins. A mutated form of XBAT31 in the RING domain (MBP-XBAT31M, H336A) was created by overlapping PCR. These fusion proteins, as well as MBP empty control, were purified with amylose agarose (BioLabs), Ni-NTA agarose (QIAGEN, Berlin, Germany), or Glutathione-Superflow resin (Takara), respectively. Ubiquitination assays were performed in reaction buffer containing 50 mM tris-HCl (pH 7.5), 20 mM ZnCl2, 5 mM MgCl2, 2 mM adenosine 5′-triphosphate (ATP), 2 mM dithiothreitol (DTT), and 20 μM MG132. After 2 hours of incubation at 30°C, the reaction was stopped by adding the SDS loading buffer. Ubiquitinated GST-ELF3 was detected by using anti-Ub (Santa Cruz Biotechnology, Dallas, TX, USA), anti-GST (Abmart), and anti-ELF3 (ABclonal) antibodies, respectively.
Glutathione superflow resin
Manufactured by Takara Bio
Sourced in United States
Glutathione-Superflow Resin is a chromatographic medium designed for the purification of glutathione S-transferase (GST) fusion proteins. The resin consists of glutathione immobilized on a cross-linked agarose support, providing a high-capacity affinity ligand for the selective capture of GST-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (5 mentions)
Chemidoc mp imaging system, by Bio-Rad (4 mentions)
Pgex 4t 1, by GE Healthcare (3 mentions)
Anti gst, by Abmart (2 mentions)
Ni nta agarose, by Qiagen (2 mentions)
Anti ub, by Santa Cruz Biotechnology (1 mentions)
Emsa kit, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 1, by Cytiva (1 mentions)
Xtractor buffer, by Takara Bio (1 mentions)
Talon metal affinity resin, by Takara Bio (1 mentions)
Anti mbp, by New England Biolabs (1 mentions)
Anti his, by Abmart (1 mentions)
12 protocols using glutathione superflow resin
1
In Vitro Ubiquitination of ELF3
2
Expression and Purification of MaNAC1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To express MaNAC1 in prokaryotes, we constructed a 35Spro‐GST‐MaNAC1 expression box. Subsequently, it was converted into E. coli strain BM Rosetta (DE3). The Glutathione‐Superflow Resin (TaKaRa) was utilized for the purification of the fusion protein. The study conducted EMSA analysis to identify the binding of MaCESA6B‐/MaCESA7‐SNBEs to MaNAC1. All specific and mutant probes were designed at 40 bp and labelled with biotin. A commercially available EMSA kit (Thermo Scientific, Rockford, IL) was used and processed according to the manufacturer's guidelines. The Light Shift chemiluminescent EMSA kit (Thermo Scientific) was utilized for detecting the bands.
3
Purification and EMSA Analysis of MYB Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For EMSA, pGEX-4T-1 (GE Healthcare) was used to generate the GST-HcMYB1 and GST-HcMYB2 expression vectors, which were transformed into E. coli strain BM Rosetta (DE3). The expression of the recombinant fusion proteins was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Following incubation at 28°C for 8 h, the fusion proteins were purified using Glutathione Superflow Resin (Clontech) according to the manufacturer’s instructions. The fragments (∼50 bp) containing putative MBE-binding sequences in the HcTPS5 and HcBSMT2 promoters were labeled with biotin. EMSA was carried out using a Light Shift Chemiluminescent EMSA Kit (Thermo Scientific) as previously described (Tan et al., 2019 (link)). The purified fusion protein was incubated with biotin-labeled DNA fragments and a 100-fold molar excess of unlabeled DNA fragments with the same sequences that were used as competitors; GST protein with labeled DNA was used as a negative control. The protein-DNA complexes were separated by 5% native polyacrylamide gel electrophoresis, detected based on chemiluminescence on a ChemiDoc MP Imaging System (Bio-Rad), and transferred onto a nylon membrane.
4
GST-tagged Protein Pulldown Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GST-tagged proteins were extracted in wash buffer A (20 mM Tris–HCl pH 8, 150 mM NaCl, and 1 mM β-mercaptoethanol) by incubation with glutathione superflow resin (Clontech, 635608), followed by washing until no protein could be detected by Bradford assay. The amount of bound protein was analysed by SDS-PAGE and staining with Coomassie dye. Volumes of bead-immobilised proteins were adjusted to ensure equal volumes of protein and made equal with unbound glutathione superflow resin, then incubated with 1 mg cell lysate in 500 µl for 1 h at room temperature. Beads were washed as for immunoprecipitation experiments and bound proteins analysed by western blotting.
5
WRKY65 Transcription Factor Binding Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The GST-BrWRKY65 expression vector was constructed with pGEX-4T-1 (GE Healthcare Life Sciences (China), Beijing, China) and then transformed into Escherichia coli strain BM Rosetta (DE3). GST-BrWRKY65 protein expression was induced by 1mM isopropyl thio-β-d -galactoside (IPTG) at 30 °C for 6 h, and the recombinant fusion protein was purified using Glutathione-Superflow Resin (Clontech, Mountain View, CA, USA) according to the manufacturer’s protocol.
The fragments of ~60 bp containing putative WRKY binding region in the promoters of BrNYC1, BrSGR2, and BrDIN1 were labeled with biotin at the 5’ end. An EMSA was performed essentially using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Rockford, IL, USA) as our previous studies described [58 (link),60 (link)]. Purified GST-BrWRKY65 fusion protein was incubated with biotin-labeled DNA fragments, and the protein-DNA complexes were separated by SDS-PAGE following detection on a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA) by the chemiluminescence method. A 100- and 1000-fold molar excess of unlabeled DNA fragments with the same or mutant sequences were used as competitors, and the GST protein alone was used as the negative control. The primers used in the EMSA assay are listed inTable S1 .
The fragments of ~60 bp containing putative WRKY binding region in the promoters of BrNYC1, BrSGR2, and BrDIN1 were labeled with biotin at the 5’ end. An EMSA was performed essentially using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Rockford, IL, USA) as our previous studies described [58 (link),60 (link)]. Purified GST-BrWRKY65 fusion protein was incubated with biotin-labeled DNA fragments, and the protein-DNA complexes were separated by SDS-PAGE following detection on a ChemiDoc™ MP Imaging System (Bio-Rad, Hercules, CA, USA) by the chemiluminescence method. A 100- and 1000-fold molar excess of unlabeled DNA fragments with the same or mutant sequences were used as competitors, and the GST protein alone was used as the negative control. The primers used in the EMSA assay are listed in
6
BrERF72 Protein Purification and EMSA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coding sequence of BrERF72 was subcloned into the pGEX-4T-1 (Amersham Biosciences) vector to produce recombinant GST-BrERF72 protein in Escherichia coli strain BM Rosetta (DE3). The induced GST-BrERF72 protein was further purified using glutathione-superflow resin (Clontech) according to the manufacturer’s protocol. The 5′ ends of synthesized oligonucleotide probes were labeled with biotin. EMSA was performed using the LightShift Chemiluminescent EMSA Kit (Thermo Scientific) as previously described7 (link),40 (link). Briefly, GST-BrERF72 protein and biotin-labeled probes were incubated together, then free and protein–DNA complexes were separated by 6% native polyacrylamide gel electrophoresis, transferred onto nylon membrane and detected by a ChemiDoc™ MP Imaging System (Bio-Rad, USA) using the chemiluminescence method. Unlabeled and mutated probes, as well as GST protein alone, were used as competitors and negative control, respectively.
7
Cloning and Purification of YcgR and BcsA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The coding regions of ycgR and bcsA(PilZ) from EC1 were amplified with the primers listed in Table S2 and cloned into GST‐tagged vector pGEX‐6P‐1 and the His‐tagged vector pCold‐SUMO, respectively. Recombinant plasmids were transformed into E. coli BL21 (DE3) for expression. Isopropyl‐1‐thio‐β‐d ‐galactopyranoside (IPTG) was added to obtain a final concentration of 0.5 mM. Cells were harvested and then resuspended in xTractor buffer (Clontech, Takara) for lysis. For GST‐tag protein, affinity purification was performed at 4°C using Glutathione‐Superflow Resin (Clontech, Takara), washed with phosphate‐buffered saline (PBS), and the target protein was eluted using gradient elution buffer (50 mM Tris‐HCl, pH 8.0) containing 33 mM reduced glutathione. For His‐tag protein, affinity purification was performed at 4°C using TALON Metal Affinity Resin (Clontech, Takara), washed with equilibration buffer (50 mM sodium phosphate, 300 mM sodium chloride, 20 mM imidazole [pH 7.4]), and the target protein was eluted with gradient elution buffer (50 mM sodium phosphate, 300 mM sodium chloride, 50/100/150/200/300 mM imidazole [pH 7.4]). All the fractions were quantified by SDS‐PAGE and Coomassie brilliant blue staining. Protein concentration was determined under A280 nm using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific).
8
Recombinant Expression and Purification of EndoS-D233Q
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The EndoS-D233Q
plasmid was previously constructed37 (link) and
was transformed into BL21 (DE3) E. coli. The transformants were cultured in 2YT broth at 37 °C. When
OD600 reached 0.8, 1 mM isopropyl β-d -thiogalactoside
was added, and the temperature was lowered to 25 °C. After overnight
growth, cells were harvested and frozen at −80 °C prior
to protein purification. Bacterial pellets were resuspended and lysed
in B-Per Bacterial Protein Extraction Reagent (Pierce) supplemented
with 10 μg/mL of DNase I. Cell lysate was centrifuged at 26000g for 20 min at 4 °C, and the supernatant was applied
to Glutathione-Superflow resin (Clontech). Samples were incubated
at 4 °C for 60 min with gentle agitation. The resin was first
washed with PBS, then 125 mM Tris, 125 mM NaCl, pH 8.0 until no protein
was detected. EndoS-D233Q was eluted with 125 mM Tris, 125 mM NaCl,
pH 8 supplemented with 10 mM reduced glutathione. Eluted protein was
concentrated to 3 mg/mL and stored at −20 °C.
plasmid was previously constructed37 (link) and
was transformed into BL21 (DE3) E. coli. The transformants were cultured in 2YT broth at 37 °C. When
OD600 reached 0.8, 1 mM isopropyl β-
was added, and the temperature was lowered to 25 °C. After overnight
growth, cells were harvested and frozen at −80 °C prior
to protein purification. Bacterial pellets were resuspended and lysed
in B-Per Bacterial Protein Extraction Reagent (Pierce) supplemented
with 10 μg/mL of DNase I. Cell lysate was centrifuged at 26000g for 20 min at 4 °C, and the supernatant was applied
to Glutathione-Superflow resin (Clontech). Samples were incubated
at 4 °C for 60 min with gentle agitation. The resin was first
washed with PBS, then 125 mM Tris, 125 mM NaCl, pH 8.0 until no protein
was detected. EndoS-D233Q was eluted with 125 mM Tris, 125 mM NaCl,
pH 8 supplemented with 10 mM reduced glutathione. Eluted protein was
concentrated to 3 mg/mL and stored at −20 °C.
9
Purification and Binding of HpWRKY44
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The N-terminus of HpWRKY44, including the WRKY domain (from 574 to 1194 bp), was inserted into pGEX-4T-1 (GE Healthcare Life Sciences (China), Beijing, China) to construct a GST-HpWRKY44 expression vector, and introduced into Escherichia coli strain BM Rosetta (DE3). GST-HpWRKY44 fusion protein was purified using Glutathione-Superflow Resin (Clontech, California, United States) after induction by 1 mM isopropyl thio-β-D-galactoside (IPTG) at 30 °C for 6 h.
LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Illinois, United States) was used to perform EMSA as previously described.33 (link),35 (link) DNA fragments, including an oligonucleotide harboring two W-box (TTGAC) motifs and the HpCytP450-like1 promoter with 74 bp containing two consensus W-box motifs were biotin-labeled at the 5′ end (primers are listed inSupplementary Table S1 ). The same or mutated probes without biotin labeling were used as the competitors. These probes were incubated with purified GST-HpWRKY44 fusion protein, and the GST protein alone was used as the negative control. After SDS–PAGE separation, protein-DNA complexes were detected using the chemiluminescence method and photos were taken on a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).
LightShift Chemiluminescent EMSA Kit (Thermo Scientific, Illinois, United States) was used to perform EMSA as previously described.33 (link),35 (link) DNA fragments, including an oligonucleotide harboring two W-box (TTGAC) motifs and the HpCytP450-like1 promoter with 74 bp containing two consensus W-box motifs were biotin-labeled at the 5′ end (primers are listed in
10
GST Fusion Protein Purification and Histone Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The glutathione S-transferase (GST) fusion proteins were expressed in Escherichia coli (Rosetta2, Novagen) at 18 °C for 10 h and were purified using glutathione superflow resin (Clontech). Then, 5 μg of GST fusion proteins and 10 μg of histone dimer, tetramer, and octamer were mixed in 200 μl of PDB buffer (50 mM HEPES-KOH, pH 7.4, 150 mM NaCl, 10% glycerol, 0.5 mM DTT, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride) and incubated at 4 °C for 2 h. After washing resins with PDB buffer three times, resins were boiled in SDS sample buffer for 5 min. The protein samples were resolved by SDS-PAGE gel, and analyzed by Coomasie blue staining. The uncropped scans of coomasie blue stained SDS-PAGE gel is shown in Supplementary Fig. 11 .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!