Affinity-purified polyclonal rabbit antibodies recognizing PDI8 were generated commercially through YenZym Antibodies, LLC (San Francisco, CA), using a truncated form of recombinant PDI8 as the antigen for both rabbit immunization and affinity purification of the antiserum. For production of the truncated PDI8 protein, a cDNA fragment encoding the central b-b’ region of PDI8 (PDI8bb’, corresponding to residues 138-377 of the PDI8 preprotein sequence) was amplified by RT-PCR using a forward primer with an NdeI site (5’-GCC TAC GCA TAT G GT TGC TCC AGA TGT GCG G-3’) and reverse primer with a BamHI site (5’-CGT GGA TCC CTA TGA GTT GAT AAA TCC CAT GAA-3’). The PDI8bb’ cDNA fragment was ligated between the NdeI and BamHI sites of the bacterial expression vector pET-15b (EMD Millipore, Billerica, MA), placing the PDI8bb’ sequence in-frame with the 6xHis-tag of pET-15b. Expression of PDI8bb’ was induced in Escherichia coli strain BL21(DE3) for 5 h at 28 °C by the addition of 0.2 mM IPTG. After induction, the E. coli cells were harvested by centrifugation and lysed using BugBuster Protein Extraction Reagent (EMD Millipore). The His-tagged PDI8bb’ protein was purified from the bacterial lysate by nickel affinity chromatography.
Pet15b
Manufactured by Merck Group
Sourced in Germany, United States, United Kingdom
PET15b is a laboratory equipment product from Merck Group. It is a plasmid DNA expression vector used for the cloning and expression of recombinant proteins in Escherichia coli. The vector contains a T7 RNA polymerase promoter, a polyhistidine (His) tag sequence, and other features that facilitate protein purification and expression.
Automatically generated - may contain errors
Lab products found in correlation
Pcoladuet, by Merck Group (9 mentions)
Pcdfduet, by Merck Group (9 mentions)
Single stranded dnas, by Integrated DNA Technologies (4 mentions)
Pacycduet, by Merck Group (3 mentions)
Pet28a, by Merck Group (2 mentions)
Ez pure plasmid prep kit, by Enzynomics (2 mentions)
Bugbuster protein extraction reagent, by Merck Group (1 mentions)
Quikchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Gibson assembly system, by New England Biolabs (1 mentions)
Hispur ni nitrilotriacetic acid nta resin, by Thermo Fisher Scientific (1 mentions)
Complete mini edta, by Roche (1 mentions)
Isopropyl β d thiogalactopyranoside, by Thermo Fisher Scientific (1 mentions)
T7 express lysy iq competent e coli, by New England Biolabs (1 mentions)
Bl21 de3 plys, by New England Biolabs (1 mentions)
Econo pac 10dg, by Bio-Rad (1 mentions)
Pet 15b, by New England Biolabs (1 mentions)
Pet15b, by Thermo Fisher Scientific (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Sanger sequencing, by Eurofins (1 mentions)
Quick ligase, by New England Biolabs (1 mentions)
31 protocols using pet15b
1
Production and Purification of PDI8 Protein
2
Cloning and Validation of Fungal Genes
3
Recombinant Giardia lamblia Protein Production
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Sequences of ENO and OCT were obtained from the genome of G. lamblia GS/M [17 (link)]. Sequence comparison between different G. lamblia assemblages and humans were done with BlastP (National Center for Biotechnology Information). Coding DNAs were generated by total gene synthesis with codon optimization for Escherichia coli (GenScript) and inserted into NdeI-XhoI cloning sites of the bacterial expression vector pET-15b (Millipore Sigma). Vectors were transformed into T7 Express lysY/Iq Competent E. coli (NEB) per the manufacturer’s protocol. Cells were grown at 37°C in LB medium with carbenicillin until they reached an optical density at 600 nm (OD600) of 0.4 to 0.6, and protein expression was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 to 3 h. Recombinant proteins were purified from bacterial lysates using HisPur Ni-nitrilotriacetic acid (NTA) resin (Thermo Scientific). Production and purification of α1-giardin and uridine phosphorylase-like protein-1 were described before [26 (link),27 (link)]. Recombinant proteins were stored at a concentration of >1 mg/ml at 4°C in a buffer of 300 mM NaCl, 25 mM HEPES, 100 mM arginine, 20 mM imidazole, 10% glycerol, 0.1% Tween 20, and protease inhibitors (Complete Mini-EDTA, Roche). Purity of the soluble recombinant proteins was analyzed by SDS-PAGE and Coomassie Blue staining.
4
Purification and Oxidation of Human CPR
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Homo sapiens cytochrome P450 reductase (CPR), lacking the 60 amino acid membrane‐binding N‐terminus, was expressed from plasmid pET15b (Millipore, Watford, Hertfordshire, UK) in Escherichia coli BL21 (DE3) pLys (New England BioLabs, Hitchin, Hertfordshire, UK), and purified by DEAE–Sepharose anion exchange chromatography followed by nickel–Sepharose affinity chromatography, as described previously40 , 41 . Prior to use, CPR was oxidized using a few grains of potassium ferricyanide, and immediately passed through an Econo‐Pac 10DG desalting column (Bio‐Rad, Hemel Hempstead, UK) to remove surplus oxidant. The oxidized CPR concentration was determined by absorption spectroscopy using an absorption coefficient (ε) of 22 mm −1·cm−1 at 454 nm 22 .
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Homo sapiens cytochrome P450 reductase (CPR), lacking the 60 amino acid membrane‐binding N‐terminus, was expressed from plasmid pET15b (Millipore, Watford, Hertfordshire, UK) in Escherichia coli BL21 (DE3) pLys (New England BioLabs, Hitchin, Hertfordshire, UK), and purified by DEAE–Sepharose anion exchange chromatography followed by nickel–Sepharose affinity chromatography, as described previously
5
In Vitro Interaction of HA-Pyk1 and Tsa1
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HA-PYK1 and TSA1 were cloned into pET15b (Millipore), and the recombinant proteins were isolated from E. coli. Recombinant HIS-HA-Pyk1 (20 μg) and recombinant HIS-Tsa1 (23 μg) were mixed in 24 mM KH2PO4/K2HPO4 (pH 7.0) and 10 mM MgSO4 (50 μl) with or without 10 mM DTT and then incubated for 10 min. The reaction mixtures were then mixed with IP buffer containing 50 mM NEM (600 μl) that included anti-HA antibody beads and incubated for 1 h with rotation. The beads were washed three times with IP buffer and then boiled in sample buffer with or without 50 mM DTT. HIS-HA-Pyk1 was detected using anti-HIS-Tsa1 antibody (Fig. 4D ) because antibody against the HIS tag had been generated during rabbit immunization rabbit with HIS-Tsa14 (link).
6
Plasmid Construction via PCR and Gibson
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Plasmids were constructed using PCR and Gibson assembly. DNA templates for repressor switch and trigger RNA expression were assembled from single-stranded DNAs purchased from Integrated DNA Technologies. The synthetic DNA strands were amplified via PCR and then inserted into plasmid backbones using 30-bp homology domains via Gibson assembly47 (link). All plasmids were cloned in the E. coli DH5α strain and validated through DNA sequencing. Backbones for the plasmids were taken from the commercial vectors pET15b (ampicillin resistance, ColE1 origin), pCOLADuet (kanamycin resistance, ColA origin), and pCDFDuet (spectinomycin resistance, CDF origin) from EMD Millipore, and the repressor DNA was inserted upstream of the T7 terminator sequence to replace their respective multiple cloning sites. GFPmut3b-ASV, GFPmut3b with an ASV degradation tag48 (link), was used as the reporter for the repressor switch plasmids, except for experiments studying GFPs with different degradation tags. In addition, the kanR mRNA toehold repressor used an mCherry reporter without a degradation tag. Sequences of elements commonly used in the plasmids are provided in Supplementary Table 1 .
7
Plasmid Construction via PCR and Gibson
8
Cloning and Expressing PeiW Protein
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Genomic DNA from M. wolfeii was obtained by extracting with a phenol-chloroform extraction procedure. The gene encoding for PeiW (psiM100p36) was amplified by PCR38 (link) and the sequence of amplified peiW was verified by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany). The insert and the Novagen vector pET-15b (Merck Group, Darmstadt, Germany) were digested with NdeI (NEB, Ipswich, MA, USA) and XhoI (NEB, Ipswich, MA, USA) and ligation of digested peiW and pET-15b with Quick ligase™ (NEB, Ipswich, MA, USA) generated the plasmid pET-15b_peiW, which was transformed into E. coli Top10. The successful transformation was confirmed by a colony PCR and by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany). Plasmid DNA was obtained by extracting with a Miniprep Kit (Pure Yield™, Promega, Madison, WI, USA), and pET-15b_peiW was transformed into E. coli DE3 BL21-AI (Life technologies, Van Allen Way Carlsbad, CA, USA). All plasmids and primers used in this study are listed in Supplementary table 1 and Supplementary table 2 respectively.
9
Construction of Synthetic RNA Circuits in E. coli
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The following E. coli strains were used in this study: BL21 DE3 (Invitrogen; F− ompT hsdSB (rB− mB−) gal dcm), BL21 AI (Invitrogen; F− ompT hsdSB (rB− mB−) gal dcm araB::T7RNAP-tetA), and DH5α (Invitrogen; endA1 recA1 gyrA96 thi-1 glnV44 relA1 hsdR17(rK− mK+) λ−).
The backbones for the plasmids used in this research were taken from the commercial vectors pET15b, pCDFDuet, pCOLADuet, and pACYCDuet (EMD Millipore). The switch RNA of the NIMPLY complex was constructed using ACTS Type II N3 and ACTS Type II N7 from previous research [46 (link)] and was constructed in pACYCDuet. All the trigger RNAs and trigger cassettes were constructed in pCDFDuet. All the antisense RNAs and antisense cassettes were constructed in pET15b. The switch RNAs of the AND gate and the NIMPLY gate were constructed in pCOLADuet. All constructs were cloned via blunt end ligation [94 (link)], Gibson Assembly [95 (link)], circular polymerase extension cloning (CPEC) [96 (link)], and/or round-the-horn site-directed mutagenesis [97 (link)]. The plasmid architecture and specific part sequences are listed inTables S3–S11 . Plasmids were constructed in E. coli DH5α and purified using the EZ-PureTM plasmid Prep Kit. Ver. 2 (Enzynomics). Plasmid sequences were confirmed by DNA sequencing after every cloning step. Plasmids were transformed through chemical transformation [98 (link)].
The backbones for the plasmids used in this research were taken from the commercial vectors pET15b, pCDFDuet, pCOLADuet, and pACYCDuet (EMD Millipore). The switch RNA of the NIMPLY complex was constructed using ACTS Type II N3 and ACTS Type II N7 from previous research [46 (link)] and was constructed in pACYCDuet. All the trigger RNAs and trigger cassettes were constructed in pCDFDuet. All the antisense RNAs and antisense cassettes were constructed in pET15b. The switch RNAs of the AND gate and the NIMPLY gate were constructed in pCOLADuet. All constructs were cloned via blunt end ligation [94 (link)], Gibson Assembly [95 (link)], circular polymerase extension cloning (CPEC) [96 (link)], and/or round-the-horn site-directed mutagenesis [97 (link)]. The plasmid architecture and specific part sequences are listed in
10
Plasmids for Yeast Two-Hybrid Assays
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Plasmids encoding enhanced green fluorescent protein (EGFP) and GAL4-DNA-binding domain (BD) fused to the C-terminus of desmoplakin (DSP C-terminus) (desmoplakin amino acid 2194 to 2871, GenBank accession No: NP_004406), with the amino acid substitution S2849G to prevent inhibitory phosphorylation, were previously described [8 (link)]. Human vimentin cDNA cloned in pDS5 was received from H. Herrmann (Heidelberg, Germany). cDNAs encoding full-length or truncated IF proteins were cloned into pAS2.1, pACT2 (Takara Bio Europe) or pACT2-URA [8 (link)] for Y2/3H assays, and into pET15b or pET23 (Merck) for expression of recombinant proteins in Escherichia coli (S1 Table ) [8 (link), 9 (link), 11 (link), 30 (link), 33 (link)]. Cloning of mouse K1-coil 1 (C1), mouse K10-C1, human K10-coil 2 to tail (C2-T), human vimentin-rod, -C1, -C2 and human desmin-rod, -C1 and -C2 into pET15b introduced an H6-tag at their N-terminus. Plasmids were generated by restriction enzyme-based cloning procedures with or without PCR amplifications. Correctness of PCR-amplified constructs was verified by sequencing.
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