Plasmids for transfection in cell culture were cloned downstream of the Cytomegalovirus (CMV) promoter of the vector pRK5-SV40-MCS. DRR1 mutants were generated by PCR mutagenesis from murine DRR1 wild-type (wt) construct in pRK5. Cloning of the murine DRR1 wt construct was previously described in [23 (link)]. The nucleotide sequences of all constructs were confirmed after cloning by Sanger sequencing. For expression of DRR1 proteins N-terminally fused to EGFP or MBP, inserts of DRR1 wt and mutants were subcloned into the vector pEGFP-C1 (Clontech, Saint-Germain-en-Laye, France) or pMAL-CR1 (New England Biolabs, Ipswich, MA, USA), respectively. Details of the cloning strategies and primer sequences are available on request.
Pmal cr1
Manufactured by New England Biolabs
Sourced in France
PMAL-cR1 is a plasmid vector designed for the expression of recombinant proteins in Escherichia coli. The vector contains the maltose-binding protein (MBP) fusion tag, which can enhance the solubility and improve the purification of the target protein. The plasmid also includes features such as a lac promoter, a multiple cloning site, and an ampicillin resistance marker.
Automatically generated - may contain errors
Lab products found in correlation
Pegfp c1, by Takara Bio (2 mentions)
Pgex 4t 1, by GE Healthcare (1 mentions)
Pmcherry c1, by Takara Bio (1 mentions)
Pcdna3 ha, by Thermo Fisher Scientific (1 mentions)
Pentr3c, by Thermo Fisher Scientific (1 mentions)
Lr recombination clonase, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
3 protocols using pmal cr1
1
Cloning and Expression of DRR1 Mutants
2
Generation of IFT46 C-terminus Antibody
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The antibodies used are listed in Supplemental Table S2. For generation of an antibody to the IFT46 C-terminus, PCR primer pair IFT46-29 and IFT46-14 was used to amplify a 0.7-kb sequence encoding IFT46 aa 105–344 from a cloned IFT46 cDNA (Hou et al., 2007 (link)). The PCR product was digested with BamHI and inserted into the BamHI site of pMAL-cR1 (New England Biolabs, Beverly, MA). Expression of this construct in Escherichia coli produced a protein in which IFT46 aa 105–344 were fused to maltose-binding protein. The fusion protein was purified by amylase affinity chromatography, and antibodies were raised against the fusion protein in guinea pigs (Covance Research Products, Denver, PA).
3
Molecular Cloning and Protein Expression
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Mouse melanophilin and syntaxin-4 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy and were verified by DNA sequencing. The sequences of the primers used are listed in Supplementary Table 2. These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG (Invitrogen), pmCherry-C1, pEGFP-C1 (Clontech), pMAL-cR1 (New England Biolabs), pGEX4T-1 (GE Healthcare Bioscience), or pCAG with a One-STrEP-Flag tag as described previously (2, 13) . Neuropeptide Y (NPY)-mCherry cDNA was generated by subcloning an mCherry cDNA into the pNPY-Venus-N1 vector. To generate recombinant adenoviruses, they were inserted into pENTR-3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen). To express exogenous protein, HEK293A cells were transfected with the plasmids using Lipofectamine 2000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses.
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