The pCS2-SENP2, pHASUMO1, pGEX-4T-SAE1/2, pGEX-2T-Ubc9, pCS2-SENP2, pCS2-SENP2M and pCS2-SENP2S DNA plasmids were described previously [16] (link). The pGEX-2T-Drp1 clone was generated by inserting a DNA fragment encoding Drp1 into the pGEX-2T vector (GE HealthCare). The SRa-HA-SUMO2, pcDNA3-HA-SUMO3 and pDsRed2-Mito clones were from Addgene or Clontech Laboratories. C3H10T1/2 and HCT116 cells and their derivatives were cultured in DMEM media with 10% fetal bovine serum and antibiotics [16] (link), [38] (link). Isolation, culture and differentiation of primary neural progenitor cells were performed as described [38] (link), [39] (link).
Pgex 2t vector
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The PGEX-2T vector is a bacterial expression vector designed for the purification of recombinant proteins. It facilitates the expression of target proteins fused to the glutathione S-transferase (GST) tag, allowing for affinity-based purification. The vector contains the tac promoter, which can be induced by IPTG, and the lacIq repressor gene, providing tight regulation of protein expression.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose bead, by GE Healthcare (3 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Pcdna3, by Thermo Fisher Scientific (2 mentions)
Pdsred2 mito, by Addgene (1 mentions)
Sodium chloride (nacl), by Nacalai Tesque (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Nacalai Tesque (1 mentions)
Gstrap ff, by GE Healthcare (1 mentions)
Tween 20, by Fujifilm (1 mentions)
Gst accept, by Nacalai Tesque (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b resin, by GE Healthcare (1 mentions)
Glutathione resin, by G Biosciences (1 mentions)
High five cells, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose affinity chromatography, by GE Healthcare (1 mentions)
Baculovirus expression system, by Thermo Fisher Scientific (1 mentions)
Pgex 4t 3 vector, by GE Healthcare (1 mentions)
Glutathione sepharose resin, by GE Healthcare (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
293fectin, by Thermo Fisher Scientific (1 mentions)
Freestyle max, by Thermo Fisher Scientific (1 mentions)
28 protocols using pgex 2t vector
1
Plasmids and Cell Culture Protocols
2
Purification and Identification of ERRα Interactors
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The pGEX-2T/ERRα wt, Δ300aa, Δ398aa or pGEX-2T vector (GE Healthcare) was transformed into the BL21 strain (RBC Bioscience) and GST fusion protein expression was induced with 0.1 mM of IPTG (Nacalai Tesque). GST or GST-ERRα was purified using GSTrap™ FF (GE Healthcare). Whole cell lysates were incubated with GST-Accept (Nacalai Tesque) overnight at 4°C. Complexes were washed three times with washing buffer (50 mM Tris-HCl, pH 7.5) (Nacalai Tesque), 150 mM NaCl (Nacalai Tesque), 0.05% Tween20 (Wako)). Proteins interacting with ERRα were eluted with the sample buffer (2% SDS, 3% glycerol, 125 mM Tris-HCl, pH 6.5), 4% 2-mercaptoethanol, and 0.0125% BPB (all from Nacalai Tesque) and detected by western blot analysis.
3
Quantifying Protein Abundance Using Recombinant Fusion Proteins
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For determination of protein abundances different recombinant fusion proteins were synthesized using a pGEX-2T vector (GE Healthcare). GSTΔhEPOR and GSTΔmEPOR consist of the complete cytoplasmic part of the respective receptor fused N-terminally to a GST tag leading to proteins of 52 214 Da mass for hEPOR and 52 309 Da for mEPOR. GSTΔJAK2 and GSTΔSTAT5 were constructed as described previously [61 (link)]. Briefly, in GSTΔJAK2 the tag is fused N-terminally to the kinase domain of murine JAK2 starting from L549 leading to a 94 572 Da fusion protein with 95% consensus to human JAK2. GSTΔSTAT5 consist of the N-terminal end of murine STAT5B starting from F332 which leads to a fusion protein of 78 432 Da with 97% consensus to hSTAT5B.
The concentrations of the recombinant proteins were determined using a BSA standard curve on a Coomassie-stained SDS-PAGE gel (SimplyBlue SafeStain, Invitrogen). Different amounts of the respective calibrators were added to the cell lysate. IP and immunoblotting was performed with the indicated total antibodies. The linear calibration curve based on the intensities of the recombinant protein was estimated with SigmaPlot (V12.5) and the endogenous signal was interpolated to calculate the corresponding number of molecules. The cell number was counted in parallel with a Neubauer improved counting chamber.
The concentrations of the recombinant proteins were determined using a BSA standard curve on a Coomassie-stained SDS-PAGE gel (SimplyBlue SafeStain, Invitrogen). Different amounts of the respective calibrators were added to the cell lysate. IP and immunoblotting was performed with the indicated total antibodies. The linear calibration curve based on the intensities of the recombinant protein was estimated with SigmaPlot (V12.5) and the endogenous signal was interpolated to calculate the corresponding number of molecules. The cell number was counted in parallel with a Neubauer improved counting chamber.
4
Recombinant Δ1–22 L-PGDS Production
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Human recombinant Δ1–22 L-PGDS was prepared as previously described (Nagata et al., 2009 (link)). The coding region of human L-PGDS without the signal sequence at the amino-terminal (amino acid residues 1–22, defining the translation initiation codon Met as 1) was cloned into the pGEX-2 T vector (GE Healthcare, Amersham, Buckinghamshire, United Kingdom) to produce a fusion protein with GST. The plasmid was transformed into E. coli BL21 (DE3) (Urade et al., 1995 (link)). The recombinant GST-fusion protein was purified using glutathione-Sepharose 4B resin (GE Healthcare), followed by digestion with thrombin. The Δ1–22 L-PGDS was further purified by gel filtration chromatography using HiLoad Superdex 75 (GE Healthcare).
5
Cloning and Purification of GST-Drn Fusion Protein
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A fragment of drn cDNA (RE70963) containing an entire open reading frame of CG45050-RC was amplified by PCR, sequenced, and subcloned into the BamHI and EcoRI sites of the pGEX-2T vector (GE Healthcare Life Sciences). A GST-Drn fusion protein was produced in Origami B (DE3) cells (Novagen) and purified using a Glutathione Sepharose 4B column. The purified GST-Drn fusion protein was used to immunize rats, and polyclonal antiserum was purified using a standard protocol.
6
Production of Anti-TSB1 Antibody
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Anti-TSB1 antibody was raised against the recombinant TSB1ΔN, TSB1 protein without chloroplast targeting signal peptide in its N-terminus, as an antigen in a rabbit (Medical & Biological Laboratories). We amplified the TSB1 DNA fragment from 1st-strand Arabidopsis cDNA by PCR using the specific primers 5′-CGGGATCCGACCCGGCCCTGTGGCAAC-3′ and 5′-CGGGATCCTCAAACATCAAGATATTTAGCCACTGTCTGAAC-3′. We cloned the TSB1 CDS of 202–1413 bp containing BamHI site at both ends into the BamHI site of the pGEX-2T vector (GE Healthcare) to express as a fusion protein with glutathione S-transferase (GST). The pGEX-2T-TSB1 was transformed into the E. coli BL21 strain. The fusion protein (GST-TSB1ΔN) was purified using the glutathione-Sepharose 4B (GE Healthcare). The TSB1ΔN protein was obtained by digestion with thrombin to cut off the GST and used for the immunization as antigen.
7
Purification and Binding of GST-Chs3 Exomer Proteins
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GST-Chs3 fragment constructs were constructed in the pGEX-2T vector (GE Healthcare) and transformed into Rosetta2 (DE3) E. coli cells (Novagen) for expression. 1 L culture was grown to ~3 OD600 in TB media at 37°C, temperature lowered to 18°C, then expression induced with 240 uM IPTG. After overnight expression, cells were harvested by centrifugation, resuspended in 50 ml PBS buffer with 1 mM DTT, and lysed by sonication. GST fusion proteins were isolated by adding 100 μl equilibrated glutathione resin (G-Biosciences) to 5 ml cleared lysate and incubating with rotation at least 2 h at 4°C. Resin was washed 3 times with 1 ml PBS+DTT and resuspended in 500 μl PBS+DTT. 10 μl of 5 mg/ml exomer protein was added and mixture was incubated ~1 h at 4°C. Resin was washed 3 times with 1 ml PBS+DTT and analyzed by SDS-PAGE and Western blot with anti-6xHis antibody (Covance).
8
Production and Purification of Integrin I Domains
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The recombinant I domains of the human integrins α1 and α2 were produced as described previously (Nykvist et al. 2000 (link)). Briefly, the α1-I domain cDNA was generated by PCR using a human integrin α1 cDNA (Briesewitz et al. 1993 (link)) as a template, cloned into the pGEX-4T-3 vector with a GST tag (GE Healthcare, USA), and the protein was expressed in E. coli. The α2-I domain cDNA was produced by PCR using a human integrin α2 cDNA (Takada and Hemler 1989 (link)) as a template, cloned into the pGEX-2T vector (GE Healthcare, USA) and the GST- α2I fusion protein was expressed in E. coli. The α11-I domain cDNA was generated similarly by PCR using a human integrin α11 cDNA (Velling et al. 1999 (link)) as a template, and then cloned into the pAcSecG2T vector (Invitrogen, USA). The α11-I domain was expressed as a GST-tagged fusion protein and secreted into cell culture media in insect High-Five cells (Invitrogen) using a Baculovirus expression system (Invitrogen). All recombinant GST-I domains were purified using glutathione-Sepharose affinity chromatography (GE Healthcare) and their purities were analyzed by SDS-PAGE.
9
Clec9A and RNF41 Protein Expression and Purification
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Clec9A ecto-domains and control proteins fused to a FLAG-tag and a biotinylation consensus sequence were expressed in mammalian Freestyle 293 F cells (Thermo Fisher) using Freestyle Max (Thermo Fisher) and purified using anti-FLAG M2 affinity gel (Sigma-Aldrich) and size exclusion chromatography (SEC) as previously described (Zhang et al., 2012 (link)). cDNA constructs encoding full-length FLAG-tagged Clec9A were subcloned into either pEF-Bos or pcDNA3.1+ (Invitrogen) for mammalian expression as previously described (Zhang et al., 2012 (link)). cDNA constructs encoding full-length and truncated versions of mouse and human RNF41 for mammalian expression (RNF41 full-length: 1-MGYD…VEEI-317; RNF41- ΔRING: 72-MRNM…VEEI-317) were subcloned into pcDNA3.1+ (Invitrogen). cDNA constructs encoding RNF41 for bacterial expression (RNF41-RBCC: 1-MGYD…VEEI-317; RNF41-BCC: 72-MRNM…VEEI-317; RNF41-CC: 135-IKHL…VEEI-317; RNF41-C: 169-DIQL…VEEI-317; RNF41-RBC: 1-MGYD…RAIR-181) were cloned into a modified pGEX-2T vector (GE Healthcare). His-tagged and untagged RNF41 proteins were expressed in mammalian Freestyle 293 F cells using 293Fectin (Thermo Fisher). GST-fusion RNF41 recombinant proteins were expressed in bacterial BL21 DE3 E. coli (Promega) and purified using Glutathione-Sepharose resin (GE Healthcare) and SEC.
10
Expression and Purification of Tfb1PH, EBNA2 Domains
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The cDNA encoding Tfb1PH (residues 1–115 of Tfb1) was cloned into the pGEX-2T vector (GE Healthcare) as previously described [39] (link). The plasmid for expressing the CBP KIX (residues 586–672 of CBP) as a His-tag fusion protein was kindly provided by Dr. Alana Schepartz (Yale University). The cDNA for EBNA2431–487 was kindly provided by Dr. S. Diane Hayward (John Hopkins University) and cloned into the pGEX-2T vector. The cDNA for the expression of EBNA2448–471 was synthetically prepared (BioCorp) and cloned into the pGEX-2T vector. The Tfb1PH and EBNA2448–471 mutants were generated using the QuikChange II site-directed mutagenesis procedure (Stratagene) starting from the wild-type sequence cloned in the pGEX-2T vector. The plasmid for expressing the LexA-EBNA2431–487 fusion protein was prepared by inserting the EcoRI-BamHI-digested PCR product generated from the corresponding pGEX-2T expression vector into the EcoRI and BamHI sites of the AB-426 vector [54] . The LexA-EBNA2431–487 mutants were generated using the QuikChange II site-directed mutagenesis procedure starting from the wild-type sequence cloned in the AB-426 vector.
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