Dsred rab7

Manufactured by Addgene

DsRed-Rab7 is a fluorescently-labeled Rab7 protein that can be used to study late endosome and lysosome dynamics in living cells. Rab7 is a small GTPase that regulates the trafficking of cargo from late endosomes to lysosomes. The DsRed fluorescent protein is fused to the Rab7 protein, allowing it to be visualized using fluorescence microscopy.

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7 protocols using dsred rab7

Plasmid Transfection in Cells

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DsRed-Rab5, DsRed-Rab7 and Flag-Rubicon plasmids were from Addgene. DsRed-LC3 plasmids were prepared in our laboratory. KSHV Flag-vBcl-2 plasmid was kindly provided by Professor Beth Levine, University of Texas Southwestern Medical Center. All plasmid constructs were confirmed by DNA sequencing. Cells were transiently transfected with the plasmids using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Zhang X., Yang Y., Liang X., Zeng X., Liu Z., Tao W., Xiao X., Chen H., Huang L, & Mei L. (2014). Enhancing Therapeutic Effects of Docetaxel-Loaded Dendritic Copolymer Nanoparticles by Co-Treatment with Autophagy Inhibitor on Breast Cancer. Theranostics, 4(11), 1085-1095.

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Reagents and Antibodies for Cell Biology

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The following reagents
and antibodies were purchased from commercial sources: Antibodies
against β-actin HRP (sc-4777), CTSL (sc-32320), GAPDH (sc-47724
HRP), normal mouse IgG (sc-2025), normal rabbit IgG (sc-2027) along
with Protein A/G PLUS-Agarose beads (sc-2003) were purchased from
Santa Cruz. Antibodies against LC3 (Cat. #12741) and Streptavidin-HRP
(Cat. #3999) were purchased from Cell Signaling Technology. Antibodies
against PSAP (A1819) and NPC2 (A5413) were purchased from Abclonal.
Protease inhibitor cocktail was purchased from Sigma-Aldrich (Cat.
P8340). Streptavidin beads, ECL Western blotting detection reagent,
and Pierce Universal nuclease were purchased from ThermoScientific.
ClarityMax Western blotting detection reagent was purchased from BioRad
(Cat. 1705062). Polyethylenimine (PEI) was purchased from Polysciences
(Cat. 4765). Inhibitors used were all purchased as follows: Bafilomycin
from CST (Cat. 54645), chloroquine diphosphate from TCI (C2301), ammonium
chloride from Fisher Scientific (A661), E64d from SeleckChem (S7393),
and Pepstatin A from Sigma (P5318). BCA assay was used for protein
concentrations. Expression plasmids for organelle markers were all
purchased from Addgene: mCherry-Sec61 (#49155), dsRed-Rab5 (#13050),
mCherry-Rab11 (#55124), dsRed-Rab7 (#12661), and Lamp1-RFP (#1817).^{40 (link)−43 (link)}

Smith M.R., Zhang L., Jin Y., Yang M., Bade A., Gillis K.D., Jana S., Bypaneni R.N., Glass T.E, & Lin H. (2023). A Turn-On Fluorescent Amino Acid Sensor Reveals Chloroquine’s Effect on Cellular Amino Acids via Inhibiting Cathepsin L. ACS Central Science, 9(5), 980-991.

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CRISPR/Cas9 Editing of TRAC Gene

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The following expression plasmids were obtained from Addgene: tf-LC3, EGFP-LC3 (#11546), mRFP-LC3 (#21075), GFP-Rab5B (#61802), EGFP-Rab6A (#49469), GFP-Rab7 (#12605), dsRed-Rab7 (#12661), dsRed-Rab11 (12679), LAMP1-RFP (#1817), mCherry-LAMP1 (#45147), mCherry-Atg5 (#13095), mCherry-p62(#55132), EGFP-Vamp7 (#42316), pEGFP-N1, and plasmids for the Sleeping Beauty transposon system, pCMV(CAT)T7-SB100 (#34879) and pSBbiGP (#60511). Plasmids for CRISPR/Cas9-mediated editing of TRAC, including pAP368 (to express TRAC gRNA-1 and EGFP reporter gene), pAP369 (to express TRAC gRNA-2 and EGFP reporter gene), and pAP370 (to express SpCas9) were obtained from Dr. Charles Gersbach’s laboratory. sgRNAs targeting TRAC were purchased from IDT; the sgRNA sequences were: AGAGTCTCTCAGCTGGTACA (sgRNA-1) and TGTGCTAGACATGAGGTCTA (sgRNA-2). PCR primers used in sequencing TRAC were TTGCTGGGGTTTTGAAGAAG (forward) and GGTTTTGGTGGCAATGGATA (reverse).

Mao M., Chang C.C., Pickar-Oliver A., Cervia L.D., Wang L., Ji J., Liton P.B., Gersbach C.A, & Yuan F. (2020). Redirecting Vesicular Transport to Improve Non-viral Delivery of Molecular Cargo. Advanced biosystems, 4(8), e2000059.

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Fluorescent Plasmid-based Autophagy Assay

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The EGFP-LC3 and DsRed-LC3 plasmids were created in our laboratory. The DsRed-Rab5, DsRed-Rab7 and Flag-Rubicon plasmids were purchased from Addgene. The KSHV Flag-vBcl-2 plasmid was kindly provided by Professor Beth Levine from the University of Texas Department of Medicine. The Rab1-37 genes in the T Vector were from Professor Jiahuai Han's Lab and sub-cloned into EGFP-C1 and DsRed-C1, respectively. All plasmids were confirmed by automated DNA sequencing. Cells were transiently transfected with the plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Zhang J., Zhang X., Liu G., Chang D., Liang X., Zhu X., Tao W, & Mei L. (2016). Intracellular Trafficking Network of Protein Nanocapsules: Endocytosis, Exocytosis and Autophagy. Theranostics, 6(12), 2099-2113.

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Fluorescent Protein-Tagged LC3 Assay

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The EGFP-LC3 and DsRed-LC3 plasmids were created in our laboratory. The DsRed-Rab7 and DsRed-Rab34 were from Addgene (Cambridge, MA). All plasmids were confirmed by automated DNA sequencing. Cells were transfected with the plasmids using Lipofectamine 2000 according to the manufacturer’s instructions.
MCF-7 cells (20,000 cells/well, 12-well plate) were seeded the day before adding the protein nanocapsules. Before the experiment, the medium was then replaced with 0.4 mL of fresh medium. After that, 50 nM FITC-labeled nanocapsules were added into cell medium and incubated at 37 °C for 12 h.

Jiang L., Liang X., Liu G., Zhou Y., Ye X., Chen X., Miao Q., Gao L., Zhang X, & Mei L. (2018). The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery. Drug Delivery, 25(1), 985-994.

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Cloning and Characterization of KIF5B Constructs

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Full-length cDNA of KIF5B was cloned into a HA-, GFP-, and FLAG-tagged expression vector, pXJ40 (E. Manser, Institute of Molecular and Cell Biology, Singapore). Constructions used in the cellular localization study were DsRed-ER (Clontech), pEYFP-Golgi (#6909-1; Clontech), mRFP-Rab5 (Vonderheit and Helenius, 2005 ; #14437; Addgene), and DsRed-Rab7 (Choudhury et al., 2002 (link); #12661; Addgene). Deletion mutants were generated by PCR using specific primers facilitated by restriction sites. For each construct, several clones were chosen and sequenced to entirety in both directions to confirm their identity. All plasmids were purified using Axygen miniprep kit for use in transfection experiments. E. coli strain DH5α was used as host for propagation of the clones.

Yi P., Chew L.L., Zhang Z., Ren H., Wang F., Cong X., Zheng L., Luo Y., Ouyang H., Low B.C, & Zhou Y.T. (2015). KIF5B transports BNIP-2 to regulate p38 mitogen-activated protein kinase activation and myoblast differentiation. Molecular Biology of the Cell, 26(1), 29-42.

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Dual-Fluorescent Protein Plasmid Transfection

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DsRed-Rab5 and DsRed-Rab7 were obtained from Addgene. KSHV Flag-vBcl-2 plasmid was kindly provided by Professor Beth Levine from Department of Medicine, University of Texas. Rab family genes in the T Vector and sub-cloned into EGFP-C1 and DsRed-C1 were kindly provided by Professor Jiahuai Han’s Lab. All the plasmids were confirmed by automated DNA sequencing. And cells were transfected with the plasmids by Lipofectamine 2000 (Invitrogen).

Sun X., Cheng C., Zhang J., Jin X., Sun S., Mei L, & Huang L. (2019). Intracellular Trafficking Network and Autophagy of PHBHHx Nanoparticles and their Implications for Drug Delivery. Scientific Reports, 9, 9585.

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