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Pdsred monomer c1

Manufactured by Takara Bio
Sourced in United States

PDsRed-Monomer-C1 is a fluorescent protein derived from the Discosoma sea anemone. It exhibits red fluorescence and can be used as a genetically encoded fluorescent label in various cell and molecular biology applications.

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7 protocols using pdsred monomer c1

1

Engineered Cardiac Receptor Constructs

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The α1A‐GFP (green fluorescent protein) and α1A‐NLSmut constructs were described previously.8 (link),16 (link) PKCδ constructs were made using a human cDNA encoding PKCδ (GenBank L07860), amplified by PCR with primers (IDT) containing Xho I (5′) and Kpn I (3′) restriction sites, and subcloned into the multicloning site of pDsRed monomer‐C1 (Clontech Laboratories, Mountain View, CA) in frame. Dominant negative PKCδ (PKCδ‐DN) was made by a K→A mutation at position 378 by site‐directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA).20 (link) The PKCδ nuclear localization mutant (PKCδ‐NLSmut) was made with a DNA minigene (IDT) containing the PKCδ NLS flanked by unique XcmI and AscI sites, but with the arginines (R) or lysines (K) replaced with alanine (A) at positions 613, 614, 615, 621, 623, 625, and 628 corresponding to the human sequence.20 (link) All DsRed‐PKCδ constructs were subcloned into the AdEasy system (Stratagene) for adenovirus production. For all experiments, cardiac myocytes were infected at a multiplicity of infection of 1000 for the α1A‐AR constructs, resulting in 2.5‐fold overexpression for the α1A‐AR,8 (link) and 5‐fold overexpression for the PKCδ constructs.
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2

Versatile expression vectors for localization

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Commercial bacterial vectors: pGEM-T Easy vector (Promega, Fitchburg, WI, USA); commercial mammalian expression vectors: pEGFP-N1 (Clontech), pDsRed Monomer-C1 (Clontech), pDsRed-Monomer-Golgi (Clontech), all utilized in localization experiments; Non-commercial mammalian expression vectors: mRFP-Dcp1a and pEYFP-TIA-1, both obtained from Paul Anderson, Harvard Medical School, Boston, MA, USA, pCR3-FL2 (it encodes the double FLAG tag, located upstream of the cloning site, and it was obtained from Ryszard Konopinski, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw, Poland).
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3

Cdc6 Derivatives Cloning and Transfection

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Open reading frames of Cdc6 derivatives were cloned into the vector pDsRed-Monomer-C1 (Clontech), unless indicated otherwise. DNA constructs were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen). The following siRNA oligonucleotides were purchased from Samchully Pharmaceutical: control GL3 siRNA, 5′-CUUACGC UGAGUACUUCGATT-3′, and Cdc6 siRNA, 5′-UAAGCCGGA UUCUGCAAGA-3′ (Lee et al., 2017 (link)). These siRNA oligonucleotides were transfected into cells using Oligofectamine (Invitrogen).
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4

UV-C Sensitivity Assay in Isogenic Cell Lines

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SV40-transformed MRC5SV1 and XP30RO (XP-V) cells were cultured in DMEM containing 10% FBS. The designations of the cell lines are abbreviated to MRC5 and XP30RO. XP30RO carrying eGFP tagged mutants of polη were described elsewhere (13 (link)). RFP-polη was created by sub-cloning the polη cDNA with XhoI and BamHI into pDsRedMonomer-C1 (Clontech). Plasmids carrying H2A-Flag and H2A (K13K15Q) -Flag were a kind gift from Lorenza Penengo and previously described (36 (link)). The eGFP tagged UBR5 was a kind gift from Darren Saunders. RNF8-Flag and RNF168-Flag expressing plasmids were a kind gift of Niels Mailand. UBR5 knock out cells were created by transfecting a plasmid encoding for a gRNA directed against exon 1 of UBR5 and CAS9-GFP. (Sigma Aldrich HS0000396905). After 24 h, the GFP positive cells were sorted and plated. After 15 days, single colonies were picked, expanded and screened by western blot for the presence of UBR5. Cells were treated with UV-C light at the doses indicated and harvested 6 h later. The MKR1 cell lines is derived from MRC5SV1 and has been described in (37 (link)).
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5

Constructing pHMGB1-EGFP and pDsRed-Atxn1-86Q

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To construct pHMGB1-EGFP, rat HMGB1 cDNA (673 bp, Rattus norvegicus, high-mobility group box 1, NM_012963, nt 29–702) was amplified from the RNA of rat cortical neurons using primers 5′-CCGCTCGAGCTGTGCCTCGCGGAGGAA-3′ and 5′-GGAATTCGTTCATCATCATCATCTTC-3′ containing XhoI or EcoRI sites and was subcloned into pEGFP-N1 (Clontech), which possesses a CAG enhancer and CMV promoter. pDsRed-Atxn1-86Q was constructed by subcloning the full-length Atxn1 cDNA fragment, amplified from pCI-Atxn1-82Q (Okazawa et al, 2002 (link)) with primers 5′-TCATCTCGAGCTATGAAATCCAACCAAGAGCGGAG-3′ and 5′-TCATGAATTCCTACTTGCCTACATTAGACCGGC-3′, between the XhoI and EcoRI sites of pDsRed-monomer-C1 (Clontech) downstream of the CMV immediate-early enhancer/promoter. During subcloning, the CAG repeat number was changed from 82 to 86, but in the final plasmid, no other sequence was changed. The pAAV1-GFP and pAAV1-HMGB1-GFP vectors were constructed by inserting HMGB1 cDNA between the XbaI and EcoRV sites of the pAAV1 vector.
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6

Cloning and Tagging of TRPM1 and NYX

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Cloning of TRPM1 (isoform C, NP_001034193.2) and NYX (NP_775591.1) from mouse retina was previously described (Agosto et al., 2014, 2018 (link)). DsRed cDNA was obtained from pDsRed-monomer-C1 (Clontech). Constructs for transfection of HEK and CHO cells were all cloned in pCDNA3.1(+) using KpnI and NotI, and consisted of free GFP or DsRed; untagged TRPM1; TRPM1 with a C-terminal 1D4 tag or N-terminal HA tag; EGFP-TRPM1 and TRPM1-EGFP fusions containing linkers GGSGG and APVAT, respectively; EGFP-NYX with EGFP behind the signal sequence (NYX[1-19]-EGFP-GGGSGGG-NYX[20-476]); NYX-EGFP (NYX-GGGSGGG-EGFP); or dsRed-NYX (Nyx[1-19]-DsRed-Nyx[20-476]). An Emerald-Sec61β construct with CMV promoter (mEmerald-Sec61-C-18) was obtained from Michael Davidson (Addgene plasmid #54249). pGrm6P-Emerald-Sec61β was constructed by cloning Emerald-Sec61β into a plasmid containing the Grm6 promoter 200-bp critical region and SV40 enhancer (Kim et al., 2008 (link)), derived from Addgene plasmid #18817, a gift from Connie Cepko, by removing the GFP, IRES, and alkaline phosphatase portions of its sequence.
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7

Mammalian Cell Expression Vectors and Reagents

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The expression vectors in mammalian cells, pAcGFP1-Actin, pAcGFP1-Tubulin, and pDsRed-Monomer-C1, were obtained from Clontech. Oligonucleotides used as PCR primers in the construction of expression plasmids for fusion proteins were custom-synthesized by Gene Design (Osaka, Japan). The WST-1 cell proliferation assay system was purchased from Takara Bio (Kyoto, Japan). Lipofectamine 2000 was obtained from Invitrogen (Carlsbad, CA). Glass-bottom dishes used for cell culture and imaging experiments were purchased from AGC Techno Glass (Shizuoka, Japan).
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