Psectag2b vector

Manufactured by Thermo Fisher Scientific

Sourced in Germany

The PSecTag2B vector is a plasmid-based expression system designed for the expression and secretion of recombinant proteins in mammalian cells. It contains a secretion signal sequence that facilitates the extracellular release of the expressed protein.

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Lab products found in correlation

Opti mem, by Thermo Fisher Scientific (2 mentions) Ni nta agarose, by Thermo Fisher Scientific (1 mentions) Pgex 6p 1 vector, by GE Healthcare (1 mentions) In fusion cloning, by Takara Bio (1 mentions) Pcdna3.1 myc his, by Thermo Fisher Scientific (1 mentions) Pfuse higg1 fc2, by InvivoGen (1 mentions) X tremegene hp dna transfection reagent, by Merck Group (1 mentions) Nonidet p 40, by Thermo Fisher Scientific (1 mentions) T5074, by Thermo Fisher Scientific (1 mentions) S271 3, by Thermo Fisher Scientific (1 mentions) Complete protease inhibitor, by Roche (1 mentions) Pcdna3.1 myc his vector, by Thermo Fisher Scientific (1 mentions) Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions) E coli dh5α cells, by New England Biolabs (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions)

8 protocols using psectag2b vector

Plasmid Constructs for Studying Lipid Signaling

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Human Pla2g2a cDNA in the pDONR221 vector was obtained from DNASU. Human Pla2g2a-Myc-His construct was obtained through a gateway reaction with the pDONR221-Pla2g2a entry vector and an engineered destination vector with CMV promoter and MycHisA tag (sPLA2-IIA-myc). Untagged human Pla2g2a was cloned into the pCDNA3.1 vector. Human PGRN and granulin-expressing constructs were previously described ^{53 (link),54 (link)}. Mouse Pla2g2a-Myc-DDK construct was obtained from Origene (m-sPLA2-IIA-myc). Mouse PGRN construct was cloned into pSecTag2B vector (Invitrogen) with an N-terminal FLAG-His tag (Flag-mPGRN). The N-terminal granulin G and F domains deleted PGRN (PGRNΔGF) (amino acids 206–593) was cloned into the pSectag2A vector with an N-terminal FLAG tag.

Du H., Yang C., Nana A.L., Seeley W.W., Smolka M, & Hu F. (2023). Progranulin inhibits phospholipase sPLA2-IIA to control neuroinflammation. bioRxiv.

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Recombinant RGMa Protein Production

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RGMa peptides were cloned in pSectag2B vector (Invitrogen, Karlsruhe, Germany) with an N-terminal His-tag. They were then transfected into HEK-293 cells for protein production. Soluble proteins were purified using Ni-NTA agarose (Invitrogen), and dialyzed against PBS.

Banerjee P., Harada H., Tassew N.G., Charish J., Goldschneider D., Wallace V.A., Sugita S., Mehlen P, & Monnier P.P. (2015). ϒ-secretase and LARG mediate distinct RGMa activities to control appropriate layer targeting within the optic tectum. Cell Death and Differentiation, 23(3), 442-453.

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Recombinant VWF A1A2A3 tridomain Protein

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Recombinant VWF A1A2A3 tridomain protein was generated using complementary DNA encoding the human VWF A1, A2, and A3 domains (amino acids Q1238-G1874) and inserted via PCR into the pSecTag2B vector (Invitrogen, CA) as described elsewhere (Auton et al., 2007 (link)). Recombinant A1A2A3 was expressed in human embryonic kidney (HEK293) cells and purified using affinity chromatography from conditioned medium. The purified A1A2A3 protein was dialyzed against 1x tris-buffered saline supplemented with 0.1% tween-20 and subjected to gel electrophoresis for verification of purity prior to experimental use (Auton et al., 2007 (link)).

Wu Y., Zeng Z., Guo Y., Song L., Weatherhead J.E., Huang X., Zeng Y., Bimler L., Chang C.Y., Knight J.M., Valladolid C., Sun H., Cruz M.A., Hube B., Naglik J.R., Luong A.U., Kheradmand F, & Corry D.B. (2021). Candida albicans elicits protective allergic responses via platelet mediated T helper 2 and T helper 17 cell polarization. Immunity, 54(11), 2595-2610.e7.

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Molecular Cloning of PSAP and PGRN

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Human PSAP cDNA in the pDONR223 vector was a gift from H. Yu (Cornell University, Ithaca, NY). His- and FLAG-tagged PSAP construct was obtained by cloning PSAP into pSectag2B vector (Invitrogen). PSAP-V5 construct was obtained through a gateway reaction with pDONR223-PSAP entry vector and pDEST6.2 destination vector (Invitrogen). GST-PSAP construct was obtained by cloning mouse PSAP into the pGEX 6p-1 vector (GE Healthcare). PGRN constructs were previously described (Hu et al., 2010 (link)). His-tagged human PSAP construct was generated by cloning human PSAP cDNA into pSectag2 vector with a 6× his tag after signal sequence. GFP-PGRN was constructed by replacing the AP sequence with GFP in the pAP5-PGRN plasmid (Hu et al., 2010 (link)). N-terminal tagged mCherry-PSAP was cloned into pCDH-puro lentiviral vector.
For the mouse PSAP shRNA targeting construct, the targeting sequence 5′-GGAACTTGCTGAAAGATAA-3′, together with short hairpins, was cloned into the BglII and XhoI sites of the pSuper vector.

Zhou X., Sun L., Bastos de Oliveira F., Qi X., Brown W.J., Smolka M.B., Sun Y, & Hu F. (2015). Prosaposin facilitates sortilin-independent lysosomal trafficking of progranulin. The Journal of Cell Biology, 210(6), 991-1002.

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NLRR1 Protein Expression and Engineering

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Human NLRR1 (NM_020873) tagged with HA at the C-terminal was cloned into the BamHI/NotI site of pcDNA3.1-myc-His (Invitrogen). This construct was used to generate other NLRR1-related plasmids. For secretion of the intact NLRR1 ectodomain, the corresponding PCR-amplified fragment of the intact extracellular region (exN1: M1-L632) was inserted into the BamHI/XhoI site of the pSecTag2B vector (Invitrogen). To fuse exN1 with human placental alkaline phosphatase (AP: NM_001632), the PCR fragment of AP (I18-G489) were inserted into the C-terminal of the pSecTag2B-exN1 vector using the In-Fusion cloning technique (Clontech). To generate the Fc chimeric proteins, intact exN1 and its deletion mutants (exN1-Fc: M1-L632; ΔLRR-Fc: N362-L632; ΔL-Ig-Fc: V516-L632) were introduced into the EcoRI site of pFUSE-hIgG1-Fc2 (Invivogen). The pEGFP-N1 plasmid was employed for EGFP-expressing cells. Expression vectors for WT ALK and ALK F1174L were kind gifts from Dr. Junko Takita (The University of Tokyo, Tokyo, Japan). The ALK R1275Q construct was generated using site-directed mutagenesis. Mission short hairpin RNA constructs, NM_020873.3-427s1c1, NM_020873.3-846s1c1, and NM_020873.3-1359s1c1, were purchased from Sigma.

Satoh S., Takatori A., Ogura A., Kohashi K., Souzaki R., Kinoshita Y., Taguchi T., Hossain M.S., Ohira M., Nakamura Y, & Nakagawara A. (2016). Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma. Scientific Reports, 6, 32682.

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Generation of AV-SNAP Fusion Protein

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The AV-SNAP fusion protein was generated by introducing the previously described sequence of AV at the SfiI and NotI restriction sites of the pMS vector system (a derivate of the pSECTag2B vector from Invitrogen), which contains the SNAP-tag sequence [8 (link), 21 (link)]. The fusion protein contains an N-terminal His₆-tag and was transiently expressed in HEK 293T cells and purified by immobilized metal-ion affinity chromatography (IMAC) from cell culture supernatant using the His-tag, as previously described [20 (link), 21 (link)]. SDS-PAGE and Coomassie Brilliant Blue staining verified the successful protein purification.

Woitok M., Grieger E., Akinrinmade O.A., Bethke S., Pham A.T., Stein C., Fendel R., Fischer R., Barth S, & Niesen J. (2020). Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes. PLoS ONE, 15(12), e0243286.

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Progranulin and proCTSD co-expression in HEK293FT

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The cDNAs for human progranulin (a generous gift from Professor Robert Farese) and proCTSD (GenScript #OHu26913) were sub-cloned into the pSecTag2B vector (ThermoFisher, #V90020). Progranulin was expressed with an N-terminal FLAG tag and proCTSD was expressed with an N-terminal His tag. HEK293FT cells (3x10⁶) were seeded into 10cm plates and grown to 60% confluency. 5μg total DNA was transfected (Opti-MEM, ThermoFisher, #31985070, X-tremeGENE HP DNA Transfection Reagent, Sigma Aldrich, #6366236001). Cells were collected forty-eight hours later and re-suspended in lysis buffer with protease inhibitor (50mM Tris pH 7.4 (Teknova #T5074), 150mM NaCl (Fisher #S271-3), 1% Nonidet P-40 (Thermo Scientific #85124), cOmplete protease inhibitor (Roche, #04693124001). Cells were lysed for one hour on ice, centrifuged at 20,000g for 20 minutes and the supernatant was collected. The protein concentration of samples was measured by BCA. Immunoprecipitation was carried out against the FLAG tag on progranulin (Sigma Aldrich, #M8823).

Butler V.J., Cortopassi W.A., Argouarch A.R., Ivry S.L., Craik C.S., Jacobson M.P, & Kao A.W. (2019). Progranulin stimulates the in vitro maturation of pro-cathepsin D at acidic pH. Journal of molecular biology, 431(5), 1038-1047.

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Recombinant ADAMTSL2 Construct Generation

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Recombinant human ADAMTSL2 constructs where generated by PCR-amplification with the Q5 Hot start high fidelity 2x master mix (NEB) and specific primer pairs to allow for restriction cloning. Cloning primers are listed in Supplemental Table S1. PCR products were amplified with the following program: 98 °C for 30 s, 34 cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 30 s/kb followed by a final extension for 2 min at 72 °C. The PCR products and the pSecTag 2B vector (ThermoFisher Scientific), which contains a signal peptide for secretion, were restriction-digested with BamHI × XhoI (NEB) and the agarose gel-purified DNA fragments were ligated with T4 DNA ligase (NEB) at 16 °C overnight. The N-terminal domains of ADAMTSL2, which include the endogenous signal peptide, were cloned in pcDNA3.1(-) Myc/His vector (ThermoFisher Scientific). Ligated plasmids were transformed in chemically competent E. coli DH5α cells (NEB, C2988J) and positive clones were identified by ampicillin resistance and verified by DNA sequencing after plasmid DNA extraction.

Kolodner P., Lukashev E.P., Ching Y.C, & Rousseau D.L. (1996). Electric-field-induced Schiff-base deprotonation in D85N mutant bacteriorhodopsin.. Proceedings of the National Academy of Sciences of the United States of America, 93(21), 11618-11621.

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