The ZIKV NS1 genes were amplified from the cDNA of infected cells and cloned into a pET-28a (+) expression vector (69864, Millipore). The cloning primers are presented in Extended Data Table 1 . Recombinant NS1 proteins were expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The proteins were then solubilized by 8 M urea and purified with a TALON Purification Kit (635515, Clontech). Murine antisera were produced via the inoculation of recombinant ZIKV NS1 with 3 boosts. Polyclonal antibodies were purified from the immunized antisera using Protein A/G agarose (20423, Thermo). The anti-V5-HRP antibody for tag detection were purchased from Invitrogen (R96125, Thermo).
Talon purification kit
Manufactured by Takara Bio
The TALON Purification Kit is a laboratory equipment product designed for the purification of recombinant proteins. It utilizes a metal-affinity resin to capture and purify proteins with a histidine tag. The kit includes pre-packed columns, buffers, and other necessary components to facilitate the purification process.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g agarose, by Thermo Fisher Scientific (4 mentions)
Express five serum free medium, by Thermo Fisher Scientific (4 mentions)
Pet28a expression vector, by Merck Group (3 mentions)
Anti v5 hrp antibody for tag detection, by Thermo Fisher Scientific (2 mentions)
Pmt bip v5 his a vector, by Thermo Fisher Scientific (2 mentions)
V4130 20, by Thermo Fisher Scientific (2 mentions)
K5130 01, by Thermo Fisher Scientific (2 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Drosophila expression system, by Thermo Fisher Scientific (1 mentions)
10 protocols using talon purification kit
1
Recombinant ZIKV NS1 Protein Production
2
Monoclonal Antibody Production and Purification of mosGCTL Proteins
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A flaviviral E protein 4G2 monoclonal antibody was produced from a hybridoma cell line (D1-4G2-4-15; ATCC). The antibodies for tags were purchased from Medical & Biological Laboratory (MBL, Japan). For antigens production, mosGCTL genes without the predicted signal sequences were amplified from A. aegypti cDNA and cloned into pET28a(+) expression vector. The cloning primers are presented in the Table S3 . The recombinant mosGCTL proteins were expressed in Escherichia coli BL21 DE3 strain, with insoluble form in inclusion bodies. The proteins were then resolved by 8 M urea and purified by TALON purification Kit (635515; Clontech). The polyclonal antibodies were produced by immunizing rabbits with recombinant mosGCTLs, including 3 boosting immunizations.
3
Expression and Purification of Mosquito Salivary Proteins
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The genes encoding A. aegypti salivary protein were cloned without signal sequences into a pMT/BiP/V5-His A vector (Cat# V4130-20, Invitrogen) for expression in Drosophila S2 cells. For immunoprecipitation assays, the human LRPPRC gene, LRPPRC truncations, PTCD1 gene, and Beclin-1 gene were cloned into a pcDNA3.1/Myc-His C vector for expression in 293T cells. LRPPRC and LRPPRC-T3 were purified from 293F cells using a Cobalt-His column. Beclin-1 was cloned into a pGEX-6P-2 vector and purified from E. coli using glutathione sepharose. The cloning primers are shown in Supplementary Table 4 . For AaVA-1 expression, the stable S2 cells were cultured in S2 Schneider’s medium in a 175 cm2 flask and then transferred into spinner flasks containing Express Five serum-free medium (Cat# 10486-025, Gibco) for protein expression. The cells were further cultured for 3 days and induced with 500 μM copper sulfate for another 4 days. The supernatant was centrifuged, filtrated, and then concentrated for AaVA-1 purification by a TALON Purification Kit (Cat# 635515, Clontech).
4
Generating Murine Polyclonal Antibodies for DENV and JEV
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To produce murine polyclonal antibodies, DENV and JEV NS1 genes were amplified from the cDNA of infected cells and cloned into a pET-28a (+) expression vector. The cloning primers are presented in Supplementary Table 2 . Recombinant NS1 proteins were expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The proteins were then solubilized using 8 M urea and purified with a TALON Purification Kit (635515, Clontech). Murine antisera were generated following 3 boosters of recombinant NS1. Polyclonal antibodies were purified from the immunized antisera using protein A/G agarose (20423, Thermo). All antibodies for tag detection were purchased from Medical & Biological Laboratory (MBL, Japan).
5
Recombinant Japanese Encephalitis Virus Envelope Protein Expression
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The JEV-E gene was amplified from the cDNA of infected cells and inserted into a pET-28a (+) expression vector (Millipore). The cloning primers are presented in Table S1. Recombinant JEV-E protein was expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The protein was then solubilized in 8 M urea and purified with a TALON Purification Kit (Clontech). Murine antiserum was produced via inoculation with recombinant E protein with three boosts, and the serum was divided and stored at −80°C until further use. The samples were separated by 12% SDS-PAGE gel, followed by electrophoretic transfer to polyvinylidene fluoride membranes and blocking and incubating membranes with primary antibodies. The following antibodies were used in these experiments: anti-V5-HRP (Invitrogen) antibodies and anti-GAPDH (Proteintech) antibodies.
6
Recombinant DENV2 NS1 Protein Expression
7
Generating Murine Polyclonal Antibodies for DENV and JEV
8
Recombinant ZIKV NS1 Protein Production
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The ZIKV NS1 genes were amplified from the cDNA of infected cells and cloned into a pET-28a (+) expression vector (69864, Millipore). The cloning primers are presented in Extended Data Table 1 . Recombinant NS1 proteins were expressed in the Escherichia coli BL21 DE3 strain in an insoluble form in inclusion bodies. The proteins were then solubilized by 8 M urea and purified with a TALON Purification Kit (635515, Clontech). Murine antisera were produced via the inoculation of recombinant ZIKV NS1 with 3 boosts. Polyclonal antibodies were purified from the immunized antisera using Protein A/G agarose (20423, Thermo). The anti-V5-HRP antibody for tag detection were purchased from Invitrogen (R96125, Thermo).
9
JEV E Protein Expression in Drosophila S2 Cells
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The JEV E gene was cloned into a pMT/BiP/V5-His A vector (Invitrogen) for expression in Drosophila S2 cells. The cloning primers are shown in Table S1. The procedure used to generate a stable cell line is described in the manual of the Drosophila expression system (Invitrogen). For JEV E expressing, the stable S2 cells were cultured in Schneider’s medium in a 225-cm2 flask. After 3 days, the medium was replaced with Express Five serum-free medium (Gibco) for protein expression. The cells were cultured for 3 days and induced with 500 µM copper sulfate for another 4 days. The supernatant was centrifuged, filtered, and then concentrated for purification using a TALON Purification Kit (Clontech). Protein purity was verified by SDS-PAGE and immunostaining with an anti-V5 mouse antibody (69 (link)).
10
Recombinant DENV2 NS1 Protein Expression
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The DENV2 NS1 gene was cloned into a pMT/BiP/myc-His A vector (modified from pMT/BiP/V5-His A, V4130-20, Invitrogen) for expression in Drosophila S2 cells. The cloning primers are shown in Supplementary Table 2 . The procedure used to generate stable cells is described in the manual of the Drosophila expression system (K5130-01, Invitrogen). NS1-expressing, stable S2 cells were then amplified in regular Schneider’s Medium in a 175- cm2 flask and transferred into spinner flasks containing Express Five serum-free medium (10486-025, Gibco) for protein expression. The cells were cultured for 3 days and induced with 500 μM copper sulfate for another 4 days. The supernatant was centrifuged, filtered, and then concentrated for purification using a TALON Purification Kit (635515, Clontech). Protein purity was verified by SDS-PAGE and immunostaining with an anti-myc mouse monoclonal antibody (M047-3, MBL, Japan).
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