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Flotillin 1

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Flotillin-1 is a membrane-associated protein that plays a role in the formation and function of lipid rafts, which are specialized microdomains in the cell membrane. It is used as a marker for lipid rafts and can be detected using immunological methods such as Western blotting or immunofluorescence microscopy.

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Lab products found in correlation

Peif2a, by Thermo Fisher Scientific (2 mentions) Cocktail of protease inhibitor, by Merck Group (2 mentions) Cd81 eat 2, by BioLegend (2 mentions) Peif2a, by Ozyme (2 mentions) Flotillin 1, by Ozyme (2 mentions) Cd63 nvg 2, by BioLegend (2 mentions) Cd9 em 04, by Abcam (2 mentions) β actin w16197a, by BioLegend (2 mentions) Ecl west pico femto, by Thermo Fisher Scientific (2 mentions) Fusion fx6 instrument, by Thermo Fisher Scientific (2 mentions) Annexin 5, by Abcam (2 mentions) β actin, by Thermo Fisher Scientific (2 mentions) Grasp65, by Thermo Fisher Scientific (2 mentions) Rab8a, by Thermo Fisher Scientific (2 mentions) Rab27a, by Thermo Fisher Scientific (2 mentions) Lc3a b, by Thermo Fisher Scientific (2 mentions) Anti mus hrp, by Thermo Fisher Scientific (2 mentions) Tsg101, by Thermo Fisher Scientific (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Rab27a, by Proteintech (1 mentions)

5 protocols using flotillin 1

1

Western Blot Analysis of Cell Lysates

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Protein lysats of cells, AB and MV were prepared in RIPA buffer containing a cocktail of protease inhibitors (Sigma). Proteins were denatured in Laemmli buffer and separated by 4-12% gradient SDS-PAGE in non-reducing (tetraspanins CD81, CD63, CD9) or reducing (all other) conditions and transferred to a nitrocellulose membrane (CD63, flotillin-1, peIF2a and CHOP; Fisher Scientific) or PVDF membrane (all other; BIO-RAD, Marnes La Coquette, France). Membranes were incubated with primary antibodies CD63 (NVG-2; 1:1000; BioLegend), CD81 (Eat-2; 1:1000; BioLegend), CD9 (EM-04; 1:1000; Abcam, Cambridge, UK), polyclonal rabbit anti-calnexin antibody (1:1000; Euromedex, Souffelweyersheim, France), β–actin (W16197A; 1:20,000; Biolegend), flotillin-1 (W16108A; 1:1000; Biolegend), peIF2a (D9G8; 1:1000; Ozyme) and CHOP (D46F1; 1:1000; Ozyme) blocked by either TBS 0.05% Tween-20 4% BSA (CD81, peIF2a, CHOP) or TBS 0.05% Tween-20 5% milk (CD9, CD63, calnexin, flotillin-1), followed by incubation with cognate HRP-conjugated secondary antibodies 1:100,000. The signals were detected with enhanced chemiluminescence substrate (ECL West Pico Femto, Fischer Scientific) on a Fusion FX6 instrument (Fisher Scientific).
Giri K.R., de Beaurepaire L., Jegou D., Lavy M., Mosser M., Dupont A., Fleurisson R., Dubreil L., Collot M., Van Endert P., Bach J.M., Mignot G, & Bosch S. (2020). Molecular and Functional Diversity of Distinct Subpopulations of the Stressed Insulin-Secreting Cell's Vesiculome. Frontiers in Immunology, 11, 1814.
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2

Western Blot Analysis of Tetraspanins

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Protein lysats of cells, AB and MV were prepared in RIPA buffer containing a cocktail of protease inhibitors (Sigma). Proteins were denatured in Laemmli buffer and separated by 4-12% gradient SDS-PAGE in non-reducing (tetraspanins CD81, CD63, CD9) or reducing (all other) conditions and transferred to a nitrocellulose membrane (CD63, flotillin-1, peIF2a and CHOP; Fisher Scientific) or PVDF membrane (all other; BIO-RAD, Marnes La Coquette, France). Membranes were incubated with primary antibodies CD63 (NVG-2; 1:1000; BioLegend), CD81 (Eat-2; 1:1000; BioLegend), CD9 (EM-04; 1:1000; Abcam, Cambridge, UK), polyclonal rabbit anti-calnexin antibody (1:1000; Euromedex, Souffelweyersheim, France), β-actin (W16197A; 1:20,000; Biolegend), flotillin-1 (W16108A; 1:1000; Biolegend), peIF2a (D9G8; 1:1000; Ozyme) and CHOP (D46F1; 1:1000; Ozyme) blocked by either TBS 0.05% Tween-20 4% BSA (CD81, peIF2a, CHOP) or TBS 0.05% Tween-20 5% milk (CD9, CD63, calnexin, flotillin-1), followed by incubation with cognate HRP-conjugated secondary antibodies 1:100,000. The signals were detected with enhanced chemiluminescence substrate (ECL West Pico Femto, Fischer Scientific) on a Fusion FX6 instrument (Fisher Scientific).
Giri K.R., Beaurepaire L.d., Jégou D., Lavy M., Mosser M., Dupont A., Fleurisson R., Dubreil L., Collot M., Endert P.V., Bach J., Mignot G., & Bösch S. (2020). Molecular and functional diversity of distinct subpopulations of extracellular vesicles from stressed pancreatic beta cells: implications for autoimmunity.
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3

Immunoblotting Protocol with Antibody Dilutions

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Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β-actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000). PAB597 is a monoclonal antibody against VP1 (Atwood et al., 1995 (link)). Secondary antibodies for Western blots include anti-Mus-HRP (Thermo A28177) and anti-Rabbit-HRP (Thermo A27036) both used at 1:10,000. Anti-rabbit-680, anti-mouse-680, anti-rabbit-800, and anti-mouse-800 (Li-Cor, Lincoln, NE) all used at 1:5,000.
Morris-Love J., O’Hara B.A., Gee G.V., Dugan A.S., O’Rourke R.S., Armstead B.E., Assetta B., Haley S.A, & Atwood W.J. (2022). Biogenesis of JC polyomavirus associated extracellular vesicles. Journal of extracellular biology, 1(5), e43.
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4

Exosome Protein Characterization by Immunoblotting

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Exosome proteins or cell lysates were blotted on a nitrocellulose membrane (5 μg) and were blocked in 5% nonfat dry milk prepared in 0.2% Tween-20 and 1× Tris Buffer Saline (TBS) and were subjected to immunoblot analysis using antibodies against Flotillin-1 (1:250, Thermo Fisher Scientific, MA, USA), TSG101 (1:250, Invitrogen, Thermo Fisher Scientific, MA, USA), ALIX, (1: 250, Cell Signaling Technology, Danvers, MA, USA), CD63, (1: 250, Cell Signaling Technology, Danvers, MA, USA), GAPDH (1: 250, DSHB, Iowa City, IA, USA), and Rab27a (1: 250, Proteintech, Chicago, IL, USA). Immune complexes were detected with appropriate goat anti-mouse (1:1000, Cell Signaling Technology, Danvers, MA, USA) and goat anti-rabbit (1:1000, Santa Cruz Biotechnology, Texas) secondary antibodies, the signal was detected using SuperSignal West Femto Maximum Sensitivity Substrate and imaged using Bio-Rad ChemiDoc XRS + System (Bio-Rad Laboratories, Hercules, CA, USA) as we described [6 ,24 ]. The Immunoblot signal was captured and analyzed via densitometric analysis on ImageJ software (NIH, Bethesda, MD, USA).
Ipinmoroti A.O., Pandit R., Crenshaw B.J., Sims B, & Matthews Q.L. (2023). Selective pharmacological inhibition alters human carcinoma lung cell-derived extracellular vesicle formation. Heliyon, 9(6), e16655.
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5

Immunoblotting Protocol with Antibody Dilutions

Check if the same lab product or an alternative is used in the 5 most similar protocols
Primary antibodies and respective dilutions for Western blots include annexin V (Abcam, Cambridge, UK ab117439, 1:1000), CD9 (Cell Signaling Techologies, Danvers, MA, CST 13174S, 1:1000), CD81 (Systems Biosciences, Palo Alto, CA, 1:1000), Flotillin-1 (CST 18634S, 1:1000), ALIX (CST 3A9, 1:1000) or (CST E6P9B, 1:1000), GRASP65 (Thermo PA3910, 1:5000), RAB8A (CST D22D8, 1:1000), RAB27A (CST D7Z9Q, 1:1000), LC3A/B (CST D3U4C, 1:1000), β-actin (CST 8H10D10, 1:1000), and PAB597 (purified, 1:2000). PAB597 is a monoclonal antibody against VP1 (Atwood et al., 1995 (link)). Secondary antibodies for Western blots include anti-Mus-HRP (Thermo A28177) and anti-Rabbit-HRP (Thermo A27036) both used at 1:10,000. Anti-rabbit-680, anti-mouse-680, anti-rabbit-800, and anti-mouse-800 (Li-Cor, Lincoln, NE) all used at 1:5,000.
Morris-Love J., O’Hara B.A., Gee G.V., Dugan A.S., O’Rourke R.S., Armstead B.E., Assetta B., Haley S.A, & Atwood W.J. (2022). Biogenesis of JC polyomavirus associated extracellular vesicles. Journal of extracellular biology, 1(5), e43.
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