Plasmids containing mCherry-Caveolin-1 (87 (link)), eGFP-LifeAct (gifts from Michael Davidson; plasmid no. 55008 and no. 54610, respectively), GFP-clathrin heavy chain (88 (link)) (gift from Stephen Royle; plasmid no. 59799), Cavin-1–mEGFP (89 (link)), Cavin-2–mEGFP (89 (link)), EHD2-mEGFP (90 (link)) (gifts from Ari Helenius; plasmid no. 27709, no. 27710, and no. 45932, respectively), GFP-Cavin-3 (27 (link)) (gift from Rob Parton; plasmid no. 68399), Dynamin-2–pmCherryN1, Eps15-pmCherryN1, Amphiphysin1-pmCherryN1, FCHo1-pmCherryC1, NECAP-pmCherryC1 (41 (link)) (gifts from Christien Merrifield; plasmid no. 27689, no. 27696, no. 27692, no. 27690, and no. 27674, respectively), GFP-Intersectin Short (91 (link)) (gift from Peter McPherson; plasmid no. 47394), Lact-C2-GFP (29 (link)) (gift from Sergio Grinstein; plasmid no. 22852), and AktPH-pmCherry (92 (link)) (gift from Moritoshi Sato; plasmid no. 67301) were obtained from Addgene. GFP-Pacsin2 (93 (link)) and CD147-GFP (94 (link)) were generated previously. Full-length constructs and all domain deletion epsin-1–mCherry constructs were generated previously (95 (link)). GFP-AP2, GFP epsin-1, and GFP-ENTH were gifts from Pietro De Camilli. DNA plasmid encoding pmKate2-f-mem (catalog no. FP186) was purchased from Evrogen.
Lact c2 gfp
Manufactured by Addgene
Sourced in United States
Lact-C2-GFP is a plasmid that expresses the C2 domain of lactadherin fused to green fluorescent protein (GFP). The C2 domain binds to phosphatidylserine, making this construct useful for the study of phosphatidylserine distribution and dynamics in cells.
Automatically generated - may contain errors
Lab products found in correlation
Gfp p4m sidm, by Addgene (2 mentions)
Lifeact egfp, by Addgene (1 mentions)
In fusion hd cloning technology, by Takara Bio (1 mentions)
Pm gfp, by Addgene (1 mentions)
Phalloidin tetramethyl rhodamine, by Merck Group (1 mentions)
Lifeact taggfp2, by Ibidi (1 mentions)
Donkey anti mouse igg fitc sc 2099, by Santa Cruz Biotechnology (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Avidin fitc, by Thermo Fisher Scientific (1 mentions)
Zeba desalt spin column, by Thermo Fisher Scientific (1 mentions)
Duramycin, by Merck Group (1 mentions)
Gfp p4m sidmx2, by Addgene (1 mentions)
Pegfp n1 α actinin 1, by Addgene (1 mentions)
Gfp c1 plcδ ph, by Addgene (1 mentions)
Pegfpc1 muscle amphiphysin 2 bin1 variant 8, by Addgene (1 mentions)
Mrfp rab7, by Addgene (1 mentions)
Mrfp rab5, by Addgene (1 mentions)
Gfp eea1, by Addgene (1 mentions)
Lamp1 rfp, by Addgene (1 mentions)
Pik93, by Bio-Techne (1 mentions)
9 protocols using lact c2 gfp
1
Fluorescent Protein Constructs for Live-Cell Imaging
2
Construction of His-Tagged Anti-GFP Nanobody
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Lact-C2-GFP (#22852) was a gift from Dr. Sergio Grinstein (Addgene, Watertown, MA, USA) [27 (link)] and pGEX6P1-GFP-Nanobody (#61838) was a gift from Dr. Kazuhisa Nakayama (Addgene, Watertown, MA, USA) [38 (link)]. The pGEX6P1-GFP-nanobody plasmid was modified by adding an amber codon (TAG) and a 6 × His-tag sequence (5′-CACCACCACCACCACCAC-3′) to the C-terminal end of the GFP open reading frame. Primers were designed to clone the His-tagged anti-GFP-nanobody sequence into a pET21b vector backbone (Table 1 ). First, Hi-Fidelity PCR was performed to linearize the anti-GFP-nanobody insert and pET21b vector backbone. Afterwards, PCR amplification insert and backbone were assembled using In-Fusion HD cloning technology (#638910) according to the manufacturer’s instructions (Clontech, Mountain View, CA, USA).
3
Quantitative Analysis of RhoA Dynamics
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Human breast cancer cell line MDA-MB-231 was purchased from ATCC. RhoA antibody (sc-418) and donkey anti-mouse IgG-FITC (sc-2099) were purchased from Santa Cruz. Duramycin, biotin and phalloidin tetramethylrhodamine (Cat#P1951) were from Sigma; biotinylated Duramycin was synthesised as described earlier (8 (link)); avidin-Texas red conjugate (A820) and avidin-FITC (Cat#43-4411) were from Life Technologies; Zeba desalt spin column (Cat#89890) was from Thermo Scientific. GFP-tagged wild-type RhoA (GFP-RhoA), constitutively active RhoA (GFP-CA-RhoA), wild-type RhoA with positive charges in the PBR mutated to Q (GFP-RhoA-PBRQ) and constitutively active RhoA with positive charges in the PBR mutated to Q (GFP-CA-RhoA-PBRQ) were prepared and used as previously described (9 (link)). Lact-C2-GFP (Cat#22852), 2PH-PLCdelta-GFP (Cat#35142) and PM-GFP (Cat#21213) were purchased from Addgene. LifeAct–TagGFP2 (Cat#60101) was purchased from Ibidi.
4
Fluorescent Tagging of HSPA1A for Localization
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To study HSPA1A’s localization, we used differentially tagged versions of the protein. Specifically, the cDNA clone containing a mouse hspA1A gene sequence, accession number BC054782, was used to generate the recombinant clones used in this study. HSPA1A was tagged with green fluorescence protein (GFP), red fluorescence protein (RFP), or myc. For expression in mammalian cells, the gene was subcloned into the pEGFP-C2, mRFP-C1, or pcDNA™3.1/myc-His(-) vectors using directional cloning after PCR amplification and restriction digest (see [22 (link),37 (link)] for complete protocol and primers used).
We used several lipid biosensors to selectively remove lipid targets using transfection (Figure 1 ). Lact-C2-GFP and Lact-C2-mCherry (PS-biosensors) were a gift from Sergio Grinstein (Addgene plasmid # 22852 and # 17274, respectively) [38 (link)]. GFP-C1-PLCδ-PH [PI(4,5,)P2 biosensor] was a gift from Tobias Meyer (Addgene plasmid # 21179) [39 (link)]. GFP-EEA1 wt [PI(3)P-biosensor] was a gift from Silvia Corvera (Addgene plasmid # 42307) [40 (link)]. GFP-P4M-SidMx2 [PI(4)P-biosensor] was a gift from Tamas Balla (Addgene plasmid # 51472) [33 (link)]. LYN11-FRB-CFP, PJ-Sac1-FKBP-mRFP, PJ-WT-FKBP-mRFP, PJ-INPP5E-FKBP-mRFP, and PJ-Dead-FKBP-mRFP were a gift from Robin Irvine (Addgene plasmid #38003, 38000, 37999, 38001, and 38002, respectively) [32 (link)].
We used several lipid biosensors to selectively remove lipid targets using transfection (
5
Plasmid Construction for Cell Biology
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Full-length EHD1 cDNA was previously described (Posey et al., 2014 (link)). Full-length EHD2-GFP was generated as described elsewhere (Doherty et al., 2008 (link)). Annexin A6–GFP and ANXA6N32 were generated as described previously (Swaggart et al., 2014 (link)). Annexin A6–mCherry and ANXA6N32-mCherry plasmids were generated by replacing the GFP tag with mCherry using BamHI and XhoI restriction sites. pEGFPC1-muscle amphiphysin II (BIN1 variant 8), GFP-C1-PLCΔ-PH, Lact-C2-GFP, pLifeAct-mTurquise2, pEGFP-N1 α-actinin1, and dysferlin-Venus were purchased from Addgene. Carboxy-terminal annexin A1-GFP, annexin A2-GFP, annexin A11-GFP, and MG53-GFP were obtained from OriGene.
6
Plasmid Cloning for Autophagy Study
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All primer sequences used in these experiments are listed in Supplementary Table 1 . MT, 2xMT, CA, CA-MT, and CA(C258S) were generated by PCR amplification of pcDNA3.1(−)-Flag-RavZ and pcDNA3.1(−)-Flag-RavZ(C258S) vectors and inserted into the N3-GFP or C1-GFP vectors using each restriction enzyme set. CA(Δα3)-MT was amplified by overlap extension PCR with C1-CFP-CA-MT used as a template, and then was inserted into the C1-GFP vector using the restriction enzyme set. GFP-AKT1-PH, GFP-Lact-C2, mRFP-Rab5 and mRFP-Rab7 were provided by Addgene (Cambridge, MA, USA). We used previously described DNA constructs for mRFP-LC3B, and mRFP-GABA RAP in this study.
7
Cloning of Molecular Constructs
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The eGFP-myc-PDK1, HA-PDK1-mCherry were cloned as described elsewhere13 (link).
mRFP-HA-PKB constructs were cloned as described elsewhere13 (link).
GFP-Lact-C2 was obtained from AddGene. Cloning of the K465A and R466A/K467A mutants of PDK1 and PHPDK1, mRFP-HA-PKB, mCherry-6xGly-GFP, mCherry-LactC2-P2A-GFP, and GRP1PH and Lyn11-FKBP2-mRFP is described in detail in the supplementary methods.
mRFP-HA-PKB constructs were cloned as described elsewhere13 (link).
GFP-Lact-C2 was obtained from AddGene. Cloning of the K465A and R466A/K467A mutants of PDK1 and PHPDK1, mRFP-HA-PKB, mCherry-6xGly-GFP, mCherry-LactC2-P2A-GFP, and GRP1PH and Lyn11-FKBP2-mRFP is described in detail in the supplementary methods.
8
Visualizing Phosphoinositide Dynamics
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All salts were from Sigma-Aldrich. PAO was from Sigma-Aldrich, and wortmannin and PIK93 were from TOCRIS Bioscience. The following plasmids were used: GFP-PH-OSBP (Balla et al., 2005 (link); gift from Tamas Balla, National Institutes of Health, Bethesda, MD), GFP-PHx2-OSH2 (Stefan et al., 2011 (link); plasmid 36095; Addgene), GFP-P4M-SidM (Hammond et al., 2014 (link); plasmid 51472; Addgene), mRFP-PH-PLCδ1, mRFP-PH-Akt, GFP-Sac2 (Nakatsu et al., 2015 (link); gift from Pietro DeCamilli, Yale University, New Haven, CT), GFP-Rab27a, GFP-Rab3a, NPY-GFP, NPY-mCherry, VAMP2-pHluorin (all gifts from Sebastian Barg, Uppsala University, Uppsala, Sweden), Lamp1-RFP (Sherer et al., 2003 (link); plasmid 1817; Addgene), GFP-Rab5, GFP-EEA1 (gifts from Pietro DeCamilli), CIBN-CAAX (Idevall-Hagren et al., 2012 (link)), GFP-CRY2-5ptaseOCRL (Idevall-Hagren et al., 2012 (link)), GFP-CRY2-iSH2 (Idevall-Hagren et al., 2012 (link)), R-GECO1 (Zhao et al., 2011 (link); plasmid 32444; Addgene), GFP-Lact-C2 (Yeung et al., 2008 (link); plasmid 22853; Addgene), EGFP-Rab4a (Rzomp et al., 2003 (link); plasmid 49434; Addgene), GFP-Rab10 (Huckaba et al., 2011 (link); plasmid 31737; Addgene), Granuphilin-GFP (Gálvez-Santisteban et al., 2012 (link); plasmid 40032; Addgene), and GFP-2xFYVEEEA1 (Petiot et al., 2003 (link)).
9
Cloning and Tagging of ER and Membrane Proteins
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Tex2 (NM_018469.5 ), TMEM55A (NM_018710.3 ), TMEM55B (NM_001100814.3 ), ALT1, ARL6IP1, E-Syt1, RTN4, and Reep5 were cloned from HeLa cDNA. The ORFs of Tex2, TMEM55B, TMEM55A, ALT1, PI4KIIα, PI4KII β, and E-syt1 were cloned into mEGFP-C1 between the BglII and SacI or Halo-C1 vector between the SacI and BamHI. The ORFs of Reep5, RTN4, and MGAT2 were cloned between NheI and SacI of Halo-N1. GST-TMEM55B was cloned into PGEX-2T vector between the BamHI and EcoRI. 14xHis-NEDD8-Tex2-SMP was cloned into 14xHis-NEDD8 vector between the BamHI and HindIII. ER-tagRFP and Lamp1-mCh were previously described (Gao et al., 2022 (link); Ji et al., 2017 (link)). GFP-Lact-C2 (22852; Addgene), CFP-C1-PLCδ-PH (21262; Addgene), GFP-P4M-SidM (51469; Addgene), mApple-Lamp1-phLuorin-N-8 (54918; Addgene), and PMRX–IP–GFP–LC3–RFP (84573; Addgene) were purchased from Addgene.
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