Mouse monoclonal anti-β-actin (Sigma), rabbit polyclonal anti-lamin B1 (MBL), rabbit polyclonal anti-DDDDK-tag (FLAG tag; MBL), mouse monoclonal anti-HA-tag (Sigma), HRP conjugated anti-mouse IgG (Sigma), HRP conjugated anti-rabbit IgG (Sigma), CF488 goat anti-mouse IgG (Biotium), and CF594 goat anti-rabbit IgG (Biotium) antibodies were used for immunoblotting and immunofluorescence. To generate NBV σA, σC, σNS, and μNS antisera, ORFs expressing these proteins were cloned into the pTrcHisA vector (Life Technologies). Plasmids were transformed into the E. coli BL21 strain, and recombinant proteins were expressed and purified. The proteins were mixed with 2% Alhydrogel adjuvant (Invivogen) and subcutaneously inoculated into ICR mice (CLEA Japan). Anti-NBV p17 antiserum was obtained by rabbit immunization with 125–138 residues of NBV p17 (Sigma).
Ptrchisa vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PTrcHisA vector is a plasmid used for the expression and purification of recombinant proteins in Escherichia coli. It contains a T7 promoter for high-level protein expression, a polyhistidine (His) tag sequence for affinity purification, and antibiotic resistance markers for selection.
Automatically generated - may contain errors
Lab products found in correlation
Icr mice, by CLEA Japan (2 mentions)
E coli top10 cells, by Thermo Fisher Scientific (2 mentions)
Mycycler gradient cycler, by Bio-Rad (2 mentions)
Gibson assembly master mix, by New England Biolabs (2 mentions)
Q5 high fidelity 2 master mix, by New England Biolabs (2 mentions)
Pgex 4t vector, by GE Healthcare (2 mentions)
Hrp conjugated anti rabbit igg, by Merck Group (1 mentions)
Alhydrogel adjuvant, by InvivoGen (1 mentions)
Hrp conjugated anti mouse igg, by Merck Group (1 mentions)
Mouse monoclonal anti β actin, by Merck Group (1 mentions)
Alhydrogel adjuvant 2, by InvivoGen (1 mentions)
13c6 15n2 lysine, by Merck Group (1 mentions)
13c6 15n4 arginine, by Merck Group (1 mentions)
Mini protean 2 cell, by Bio-Rad (1 mentions)
Halolink resin, by Promega (1 mentions)
9 protocols using ptrchisa vector
1
Generating Antibodies and Antisera
2
Generating Antiserum Against Viral Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate antiserum against strain MB σC, the σC coding region of the MB S1 gene was cloned downstream of sequences encoding poly- histidine (His) tag in the pTrcHisA vector (Life Technologies). The His-σC fusion protein expressed in BL21 cells (Takara) was purified from the soluble fraction using His-Select R Nickel Affinity Gel (Sigma) according to the manufacturer’s instructions. The His-σC fusion protein was mixed with Alhydrogel adjuvant 2% (InvivoGen) according to the manufacturer’s instructions, and ICR mice (CLEA Japan) were immunized and boosted with the protein-adjuvant mixture to generate σC-specific serum. Antiserum was obtained 4 weeks after administration of the last booster. To generate antiserum against strain MB, virions were mixed with Alhydrogel adjuvant 2% according to the manufacturer’s instructions, and mice were immunized and boosted with the virus-adjuvant mixture. Antiserum was obtained 4 weeks after administration of the last booster.
3
Stable Isotope Labeling of Ras Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
13C6-15N2-lysine (+8 Da) and 13C6-15-N4-arginine (+10 Da) were obtained from Sigma. His-tagged, wild-type KRAS4B and NRAS plasmids were kindly provided by Ignacio Rubio (Institute of Molecular Cell Biology, University of Jena). Wild-type, full-length human HRAS and KRAS4A sequences were sub-cloned into the pTrcHis A vector (Invitrogen) from plasmids described in [23 (link), 24 (link)] and sequence verified.
+ Expand
13C6-15N2-lysine (+8 Da) and 13C6-15-N4-arginine (+10 Da) were obtained from Sigma. His-tagged, wild-type KRAS4B and NRAS plasmids were kindly provided by Ignacio Rubio (Institute of Molecular Cell Biology, University of Jena). Wild-type, full-length human HRAS and KRAS4A sequences were sub-cloned into the pTrcHis A vector (Invitrogen) from plasmids described in [23 (link), 24 (link)] and sequence verified.
4
Cloning and Characterization of clbH Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Linearized pTrcHisA vector was PCR amplified from pTrcHisA vector (Invitrogen) using primers shown in Supplementary Table 4 . Full-length clbH, truncated clbH (clbH-C-A2-PCP), and two adjacent genes clbH and clbI (clbH-clbI) were PCR amplified from E. coli CFT073 genomic DNA (ATCC) using primers shown in Supplementary Table 4 . PCR reactions (20 μL) contained 10 μL Q5 High-Fidelity 2× Master Mix (New England Biolabs), 1 ng of DNA template, and 500 pmoles of each primer. Thermocycling was carried out in a MyCycler gradient cycler (Bio-rad) using the following condition: denaturation for 1 min at 98 °C, followed by 35 cycles of 10 sec at 98 °C, 30 sec at 72 °C, 3 min for clbH (2 min for clbH-C-A2-PCP and 4 min for clbH-clbI) at 72 °C, and a final extension of 5 min at 72 °C.
Gibson assembly reactions (10 μL) contained 100 ng linearized pTrcHisA vector, 3-fold of molar excess purified PCR products of clbH, clbH-C-A2-PCP, or clbH-clbI, and 5 μL of 2× Gibson Assembly Master Mix (New England Biolabs). The mixtures were incubated at 50 °C for 15 min and used to transform 50 μL of chemically competent E. coli TOP10 cells (Invitrogen). The identity of the assembled plasmids was confirmed by sequencing.
The pTrcHisA-clbH, -clbH-C-A2-PCP, and -clbH-clbI plasmids were electroporated into electrocompetent E. coli DH10B BACpksΔclbDtoMΔclbOtoQ and stored at −80 °C as frozen LB/glycerol stocks.
Gibson assembly reactions (10 μL) contained 100 ng linearized pTrcHisA vector, 3-fold of molar excess purified PCR products of clbH, clbH-C-A2-PCP, or clbH-clbI, and 5 μL of 2× Gibson Assembly Master Mix (New England Biolabs). The mixtures were incubated at 50 °C for 15 min and used to transform 50 μL of chemically competent E. coli TOP10 cells (Invitrogen). The identity of the assembled plasmids was confirmed by sequencing.
The pTrcHisA-clbH, -clbH-C-A2-PCP, and -clbH-clbI plasmids were electroporated into electrocompetent E. coli DH10B BACpksΔclbDtoMΔclbOtoQ and stored at −80 °C as frozen LB/glycerol stocks.
5
Cloning and Characterization of clbH Gene
6
Cloning and Purification of VviDREBA1 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The full VviDREBA1s [GenBank Accession No. MF445007 (VviDREBA1-1), MF445008 (VviDREBA1-6) and MF445009 (VviDREBA1-7)] open reading frames (ORFs), including stop codons, were amplified by RT-PCR using the primers included in the Supplementary Table S1 . The forward VviDREBA1-7 and VviDREBA1-1 primers contained a BamHI site, and the forward VviDREBA1-6 primer contained a XhoI site. The three reverse primers contained an EcoRI site. The resulting fragments digested with their respective restriction enzymes were cloned into the pTrcHisA vector, which contains an N-terminal His6 (Invitrogen, Carlsbad, CA, United States), previously digested with the same enzymes, and transformed into BL21-CodonPlus (DE3)-RIL competent cells. The induction and purification of recombinant proteins were performed according to Romero et al. (2008) (link). The purified fusion proteins were concentrated as described by Romero et al. (2016) (link). Protein analyses were performed on 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) using Mini-Protean II Cell (Bio-Rad) equipment as described by Rosales et al. (2014) (link). Western blots were probed with antibodies and conditions previously described by Romero et al. (2016) (link).
7
Mammalian Expression Plasmids and Constructs
8
Purification of C2AB and SNARE Complexes
9
Purification of Synaptotagmin Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Constructs encoding syt-1 C2AB (a.a. 96–421) and syt-17 C2AB (a.a. 152–474) were expressed as GST fusion proteins (pGEX-4T vector, GE) in E. coli, purified via glutathione-Sepharose affinity chromatography, and cleaved with thrombin in 100 mM KCl, 25 mM HEPES pH 7.4, 5% glycerol. HaloTag constructs were assembled by overlap extension PCR and subcloned into pTrcHis A vector (ThermoFisher) to yield N-terminal His6-HaloTag-syt constructs. These constructs were expressed in E. coli, purified via nickel-NTA chromatography, and eluted in Hisg-tag elution buffer (500 mM imidazole, 400 mM KCl, 25 mM HEPES pH 7.4, and 10% glycerol). Full-length syt-17 was purified by affinity chromatography of E. coli lysates using HaloLink resin (Promega) and eluted by cleavage with TEV protease in 100 mM KCl, 25 mM HEPES pH 7.4, 5% glycerol, 1% Triton X-100 with 2 mM DTT.
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!