To construct the mKate2-TAPP1-PH, BamHI-mKate2 and TAPP1-PH-Not1 were prepared by PCR with mKate2-P2A-APEX2-TAPP1-PH (Addgene number; #67662) as a template. These PCR products were inserted in pRK5 vector using In-Fusion technique (Takara). pTag-BFP-C-h-Rab5a-c-Myc (#79801), GFP-Rab7a (#61803), LAMP1-mGFP (#34831), and pEGFPN1-human Dynamin K44A (#22197) were obtained from Addgene. FRET-based RhoA biosensor is originally from Dr. Matsuda (Osaka University)39 (link). mCherry-tagged ARAP3 and ARAP3-R982A were generated by fusing red fluorescent protein mCherry into the N-terminal of the ARAP3 templates34 (link). To construct the EGFP-ARAP3-PH(X), each PH domain35 (link) and EGFP sequences were prepared by PCR and inserted in pRK5 vector using In-Fusion technique. VN173-tagged TAPP1 was constructed by inserting PCR fragments of VN173 and TAPP1 PH domain in pRk5 vector. VC155-tagged ARAP3-PH(X) constructs were generated by Xba1/BamH1 replacement of EGFP in EGFP-ARAP3-PH(X) with a VC155 including Xba1/BamH1 site.
Ptag bfp c h rab5a c myc
Manufactured by Addgene
PTag-BFP-C-h-Rab5a-c-Myc is a plasmid construct that expresses a fusion protein consisting of a Blue Fluorescent Protein (BFP) tag and the human Rab5a protein with a C-terminal c-Myc tag. The construct is designed for the expression and visualization of the Rab5a protein in mammalian cells.
Automatically generated - may contain errors
Lab products found in correlation
In fusion technique, by Takara Bio (1 mentions)
Gfp rab7a, by Addgene (1 mentions)
Mkate2 p2a apex2 tapp1 ph, by Addgene (1 mentions)
Lamp1 mgfp, by Addgene (1 mentions)
Pcdna4 vps34 flag, by Addgene (1 mentions)
Mcherry rab5ca q79l, by Addgene (1 mentions)
Kod plus mutagenesis kit, by Toyobo (1 mentions)
2 protocols using ptag bfp c h rab5a c myc
1
Fluorescent Biosensor Cloning Protocol
2
Rab5-Mediated Vesicle Dynamics Protocol
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pET-GST-R5BD-hRABEP1 was purchased from Vector Builder. Rab5a-pmCherryC1 was a gift from Christien Merrifield (Addgene plasmid #27679; http://n2t.net/addgene:27679 , accessed on 31 May 2019; RRID:Addgene_27679) [41 (link)]. pcDNA4-Vps34-Flag was from Qing Zhong (Addgene plasmid #24398; http://n2t.net/addgene:24398 , accessed on 12 November 2018; RRID:Addgene_24398) [42 (link)]. mCherry-Rab5CA(Q79L) was from Sergio Grinstein (Addgene plasmid #35138; http://n2t.net/addgene:35138 , accessed on 26 April 2018; RRID:Addgene_35138) [43 (link)]. TagRFP-T-EEA1 was from Silvia Corvera (Addgene plasmid #42635; http://n2t.net/addgene:42635 , accessed on 9 November 2017; RRID:Addgene_42635) [44 (link)]. pTag-BFP-C-h-Rab5a-c-Myc was from James Johnson (Addgene plasmid #79801; http://n2t.net/addgene:79801 , accessed on 9 November 2017; RRID:Addgene_79801) [45 (link)]. RFP-Rab5 140 and 180 mutants were generated with primers (5′-GCAGCAGCTGTTGACTTCCAGG-3′ and 5′-ATTTGCTAAGTCAGCTTTGTTTCCTGAC-3′) and (5′-GCAGCGCTGCCAAAGAATGAAC-3′ and 5′-AGCTATTGCCATAAATATTTCATTTACATTCATTG-3′), respectively, using a KOD-plus mutagenesis kit (Toyobo, Osaka, Japan).
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