AgBR1 and NeSt1 were previously cloned in-frame into the pMT-Bip-V5-His tag vector (Invitrogen) [21 ] and recombinant proteins expressed and purified using the Drosophila Expression System (Invitrogen). AgBR1 and NeSt1 recombinant proteins were purified from the supernatant by TALON metal affinity resin (Clontech) and eluted with 150 mM imidazole. The eluted samples were filtered through a 0.22-μm filter and concentrated with a 10-kDa concentrator (Sigma-Aldrich) by centrifugation at 4°C and washed and dialyzed in PBS. Recombinant protein purities were assessed by SDS-PAGE, and then quantified using the Pierce BCA Protein Assay kit (Thermo Scientific). To generate rabbit sera against recombinant proteins, rabbits were immunized subcutaneously with 100 μg of recombinant proteins in complete Freund’s adjuvant and boosted three times at 2 week-intervals with 50 μg of recombinant proteins in incomplete Freund’s adjuvant. Rabbits were euthanized 2 weeks after the final boost and serum was collected by cardiac puncture. Reactivity to recombinant proteins was verified by immunoblot.
Pmt bip v5 his tag vector
Manufactured by Thermo Fisher Scientific
The PMT-Bip-V5-His tag vector is a laboratory tool used for the expression and purification of recombinant proteins. It contains a Protein A-Binding Immunoglobulin-Binding (PMT-Bip) tag and a V5 and 6xHis tag, which can be used for affinity purification and detection of the expressed protein.
Automatically generated - may contain errors
Lab products found in correlation
Drosophila expression system, by Thermo Fisher Scientific (5 mentions)
10 kda concentrator, by Merck Group (4 mentions)
Talon metal affinity resin, by Takara Bio (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bca protein estimation kit, by Thermo Fisher Scientific (3 mentions)
5 protocols using pmt bip v5 his tag vector
1
Recombinant Protein Purification and Antibody Generation
2
Recombinant AgBR1 Protein Purification
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AgBR1 with a TEV tag was cloned in frame into the pMT-Bip-V5-His tag vector (Invitrogen) and recombinant proteins expressed and purified using the Drosophila Expression System (Invitrogen). AgBR1-TEV-V5-His was purified from the supernatant by TALON metal affinity resin (Clontech) and eluted with 150 mM imidazole. The eluted samples were filtered through a 0.22 μm filter and concentrated with a 10-kDa concentrator (Sigma-Aldrich) by centrifugation at 4 °C, and washed and dialyzed in PBS. Recombinant protein purities were assessed by SDS-PAGE, and then quantified using the Pierce BCA Protein Assay kit (Thermo Scientific). TurboTEV Protease (Accelagen Inc.) was used to cleave the tags from AgBR1-TEV-V5-His following the manufacturer’s instructions to obtain untagged AgBR1 protein for immunizations.
3
Ixofin3D Recombinant Protein Expression
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Ixofin3D was cloned in-frame into the pMT-Bip-V5-His tag vector (Invitrogen, CA) using ixofin3D_DESF and ixofin3D_DESR primers listed in Table S1 and recombinant protein expressed and purified using the Drosophila Expression System (Invitrogen, CA) as described earlier [22] (link). Recombinant protein purity was assessed by SDS-PAGE, and quantified using the BCA protein estimation kit (Thermoscientific, IL).
4
Recombinant Protein Purification and Antibody Generation
5
Recombinant Protein Purification and Antibody Generation
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AgBR1 (AAEL001965), SP (AAEL003600) and D7Bclu (AAEL006417) were cloned in-frame into the pMT-Bip-V5-His tag vector (Invitrogen, CA) and recombinant proteins expressed and purified using the Drosophila Expression System (Invitrogen, CA) as described earlier14 (link). AgBR1, SP and D7Bclu were purified from the supernatant by TALON Metal Affinity Resin (Clontech, CA) and eluted with 150 mM imidazole. The eluted samples were filtered through a 0.22-μm filter and concentrated with a 10-kDa concentrator (Sigma-Aldrich, MO) by centrifugation at 4°C, washed and dialyzed against PBS. Recombinant protein purities were assessed by SDS-PAGE and quantified using the BCA protein estimation kit (Thermo scientific, IL). The PCR primer sequences for cloning are listed in Supplementary Table 6 .
To generate rabbit sera against recombinant proteins, rabbits were immunized subcutaneously with 80–150 μg of recombinant proteins in complete Freund’s adjuvant and boosted twice at every 2 weeks with 80–150 μg of recombinant proteins in incomplete Freund’s adjuvant. Rabbits were euthanized and sera were obtained by cardiac puncture 2 weeks after the final boost. Reactivity to recombinant proteins was examined by immunoblot and ELISA.
To generate rabbit sera against recombinant proteins, rabbits were immunized subcutaneously with 80–150 μg of recombinant proteins in complete Freund’s adjuvant and boosted twice at every 2 weeks with 80–150 μg of recombinant proteins in incomplete Freund’s adjuvant. Rabbits were euthanized and sera were obtained by cardiac puncture 2 weeks after the final boost. Reactivity to recombinant proteins was examined by immunoblot and ELISA.
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