Tgn46

Manufactured by Thermo Fisher Scientific

Sourced in Israel

TGN46 is a laboratory instrument designed for the detection and quantification of specific biomolecules in samples. It utilizes advanced technology to provide accurate and reliable results for researchers and scientists. The core function of TGN46 is to enable precise analytical measurements, supporting a wide range of applications in various scientific fields.

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5 protocols using tgn46

Immunofluorescence Staining of Tissue Sections

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Tissue specimens were washed in cold PBS, fixed in 10% formalin buffered saline for 2 hr, followed by incubation in 30% sucrose overnight. Tissue specimens were embedded in optimal tissue cutting medium and 6-µm-thick section were cut. Sections were rinsed in PBS followed by antigen retrieval in 0.05% citraconic anhydride (Sigma-Aldrich, St Louis, MO), permeabilized in 0.05% Triton X-100 or 0.05% saponin (TGN46) (Sigma-Aldrich, St Louis, MO), washed in PBS, and blocked in 5% goat serum (Sigma-Aldrich, St Louis, MO). Sections were incubated with primary antibodies toward EEA1, Rab7 (Cell Signaling Technology, Beverly, MA), Lysozyme, TGN46 (Thermo Fisher Scientific, Waltham, MA), Rab3D (Synaptic Systems, Goettingen, Germany), LAMP1, Calnexin (Abcam, Cambridge, United Kingdom), or VAMP-8 (gift from Prof Burton Dickey, University of Texas) at 4°C overnight, rinsed with PBS and incubated with goat anti rabbit or goat anti chicken Alexa Fluor conjugated secondary antibodies (Thermo Fisher Scientific, Waltham, MA) at room temperature for 1 hr. Sections were counterstained with DAPI and imaged using an Axioskop 2 microscope, an LSM700 Axio Examiner Z1 confocal microscope, or an LSM900 with Airyscan 2 microscope (Carl Zeiss Microscopy, Thornwood, NY). Acquired images were analyzed using Zen (Carl Zeiss Microscopy, Thornwood, NY) and Imaris (Bitplane, Belfast, Great Britain) software.

Gustafsson J.K., Davis J.E., Rappai T., McDonald K.G., Kulkarni D.H., Knoop K.A., Hogan S.P., Fitzpatrick J.A., Lencer W.I, & Newberry R.D. (2021). Intestinal goblet cells sample and deliver lumenal antigens by regulated endocytic uptake and transcytosis. eLife, 10, e67292.

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Golgi Localization of Ion Channels

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To evaluate the localization of our proteins relative to Golgi apparatus, cells were treated with cell light Golgi-GFP (Life Technologies, Eugene, OR; 5 μl/10000 cells) 48 hours before fixation, simultaneously of cells starvation. Cells were fixed with paraformaldehyde 4% for 20 minutes, permeabilized with triton 0.5% and blocked for 30 minutes with BSA 3%. The cells are then incubated overnight with primary antibodies (Kv10.1 1/50, Santa Cruz Biotechnology, Inc., Heidelberg, Germany; Orai1 1/200, Sigma Aldrich, Saint-Quentin-Fallavier, France; SPCA2 1/100, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), washed with PBS, incubated 1 hour with secondary antibodies conjugated with fluorophores (anti-mouse Alexa 546, 1/200, Life Technologies, Eugene, OR; anti-rabbit Alexa 549, 1/200, Thermo Fisher Scientific, Rockford, IL) and counterstained with DAPI to visualize the nuclei. Analyses were performed on a LSM 780 confocal microscope (Carl Zeiss) and analyzed with ZEN 2012 software. For immunofluorescence assay on human tissue, samples were stained with anti-Kv10.1 (1/50, Alomone Labs, Jerusalem, Israel) and TGN46 (1/100, Thermofisher Scientific).

Peretti M., Badaoui M., Girault A., Van Gulick L., Mabille M.P., Tebbakha R., Sevestre H., Morjani H, & Ouadid-Ahidouch H. (2019). Original association of ion transporters mediates the ECM-induced breast cancer cell survival: Kv10.1-Orai1-SPCA2 partnership. Scientific Reports, 9, 1175.

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Immunofluorescence and Western Blot Protocols

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For immunofluorescence the following primary antibodies were used: GFP (Santa Cruz, sc-9996) and TGN46 (Thermo, pa 1-1069). Secondary antibodies used are the following: Alexa 488 (Invitrogen, a21202 and a21206), Alexa 594 (Invitrogen, a21442) and Alexa 647 (Invitrogen, a281883). For Western Blot the following primary antibodies were used: GFP (Santa Cruz, sc-9996), actin (Abcam, ab6276), Ykt6 (Abcam, ab236583), LC3B (Cell Signaling, #2775), p62
(Sigma, p0067), STX17 (Sigma, hpa001204), SNAP29 (Abcam, ab138500) Na+/K+ ATPase (Sigma, 06-172-I), alpha/beta-Tubulin (Cell Signaling, 2148S), and STX7 (Bethyl, A304-512A).
Secondary antibodies used are the following: IRDye680 (Fisher, 925-68070) and IRDye800
(Fisher, 925-32211). Secondary antibodies used are the following: IRDye680 (Fisher, 925-68070) and IRDye800 (Fisher, 925-32211).

McGrath K., Dergai M., Agarwal S., Chung D., Rossum D.B.v., Shevade A., Kuchin S., Zaichick S., Savas J.N., Fasshauer D., & Caraveo G. (2020). A conformational switch driven by phosphorylation regulates Ykt6 activity in macroautophagy.

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LRRK2 Localization Modulation by Rab7L1

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HEK 293FT cells were seeded at 1×10⁴ cells per well in a 96-well Matrigel coated plate. Cells were transfected by reverse transfection with pooled ON-TARGETplus siRNA against ARHGEF7, CSNK1A1or non-targeting control (Thermo) at a final concentration of 25 nM. Transfection was carried out according to the manufacturer’s protocol, using DharmaFECT 1 transfection reagent (Thermo). 24 hrs after siRNA transfection, cells were further transfected with 3xFLAG -LRRK2 and 2xmyc-GUS or 2xmyc-Rab7L1 WT or 2xmyc-Rab7L1 Q67L mutant plasmids using Lipofectamine-2000 (Life Technologies) reagent. 48 hrs after siRNA transfection and 24 hrs after plasmids transfection cells were fixed with 4% paraformaldehyde containing 1 µg/ml Hoechst and stained for endogenous TGN46 (AbD Serotec), FLAG (Sigma) and myc (Roche) antibodies. Plates were imaged at 20x objective on high throughput Cellomics VTI arrayScanner and analyzed using Spot Detector bioapplication for % of cells with LRRK2 and TGN46 positive spots from total number of LRRK2 transfected cells. Three independent experiments were performed with 6 wells per sample with the minimum of 1000 cells/well. siRNAs samples for Rab7L1, ARHGEF7, or CK1α were compared to NTC within families (GUS, Rab7L1 WT, or Rab7L1 Q67L) by two way ANOVA, Tukey’s multiple comparison test.

Chia R., Haddock S., Beilina A., Rudenko I.N., Mamais A., Kaganovich A., Li Y., Kumaran R., Nalls M.A, & Cookson M.R. (2014). Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7. Nature communications, 5, 5827.

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LRRK2 Localization Modulation by Rab7L1

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