Fluorescent secondaries

Manufactured by LI COR

Fluorescent secondaries are laboratory reagents used in immunoassays and other fluorescence-based techniques. They are designed to bind to primary antibodies, allowing for the detection and visualization of target proteins or other biomolecules. These secondary antibodies are conjugated with fluorescent dyes, enabling the signal to be captured and quantified using appropriate instrumentation.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Odyssey system and software, by LI COR (1 mentions) Immobilon fl, by Merck Group (1 mentions) Gapdh, by Merck Group (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Nupage sample buffer, by Thermo Fisher Scientific (1 mentions) A2228, by Merck Group (1 mentions) Intercept blocking buffer, by LI COR (1 mentions) Intercept protein free blocking buffer, by LI COR (1 mentions) Ripa buffer, by Teknova (1 mentions) G4171, by Merck Group (1 mentions) G8795, by Merck Group (1 mentions) Semi dry transfer apparatus, by Bio-Rad (1 mentions) Mes buffer, by Thermo Fisher Scientific (1 mentions)

2 protocols using fluorescent secondaries

Myelin-enriched Proteins Analysis in Mouse Brain

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In a separate cohort of mice after one month of hypoperfusion (n = 8) or a sham (n = 5) procedure, after decapitation, the brain was rapidly removed, the cerebellum discarded and the remaining brain frozen in liquid nitrogen. Myelin-enriched fractions were prepared by sucrose density centrifugation [29] (link) and the total protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, UK). Proteins were separated by Bis-Tris 4–12% SDS-PAGE (NuPage® Novex®, Life Technologies) and transferred onto PVDF membrane (Immobilon-FL, Millipore). Immunobloting was performed using the Odyssey Infrared Imaging System (LiCor Biosciences, Lincoln, NE, USA). Membranes were blocked 1 hour at room temperature in Odyssey blocking buffer (diluted 1∶1 with phosphate-buffered saline), washed in phosphate-buffered saline–Tween (phosphate-buffered saline with 0.1% Tween) and incubated over-night at 4°C with MBP (1∶10.000, Millipore). After gentle washing, membranes were then incubated 1 hour at room temperature with GAPDH (1∶14.000, Sigma) which was used as a loading control. Membranes were then incubated for 45 minutes with the appropriate fluorescent secondaries (1∶3000, LiCor Biosciences). The western blots were analysed using the LiCOR Bioscience Odyssey system and software. The four MBP isoforms were analysed together and normalized to GAPDH.

McQueen J., Reimer M.M., Holland P.R., Manso Y., McLaughlin M., Fowler J.H, & Horsburgh K. (2014). Restoration of Oligodendrocyte Pools in a Mouse Model of Chronic Cerebral Hypoperfusion. PLoS ONE, 9(2), e87227.

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Western Blot Analysis of Protein Expression

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Brain and liver homogenates were generated as detailed above. BMDMs cultured in 96-well format were washed 2x with PBS and then collected in 25uL 1x CST Buffer (Cell Signaling, #9803S) supplemented with PMSF. 1mg/mL tissue/cell lysates were prepared for SDS-PAGE by diluting with 4X NuPage Sample Buffer (Invitrogen #NP007), 10X NuPage Reducing Buffer (Invitrogen #NP009), and RIPA Buffer (Teknova #R379). Samples were boiled for 5 min, and then run on a 4–12% Bis-Tris gel (Invitrogen #NP0322) in MES buffer (Invitrogen #NP0002), 120V for 1.5 hours. Gel was then transferred on the Bio-Rad semi-dry transfer apparatus mixed molecule weight program onto nitrocellulose. Transferred blot was blocked with Intercept protein-free blocking buffer (Licor, #927–80001). Blot was then probed for GCase (Sigma #G4171, 1:1000) and GAPDH (Sigma #G8795, 1:2500) for liver and brain tissues or Cathepsin D (Abcam #ab78552, 1:1000) and beta-Actin (Sigma #A2228, 1:3000) for BMDM cell lysates and detected via fluorescent secondaries (Licor) while incubating in LI-COR Intercept Blocking Buffer. After washing, blot was imaged and quantified on the Odyssey scanner, and analyzed in Prism by one-way ANOVA and Dunnett’s multiple comparison test.

Logan T., Simon M.J., Rana A., Cherf G.M., Srivastava A., Davis S.S., Yoon Low R.L., Chiu C.L., Fang M., Huang F., Bhalla A., Llapashtica C., Prorok R., Pizzo M.E., Calvert M.E., Sun E.W., Hsiao-Nakamoto J., Rajendra Y., Lexa K.W., Srivastava D.B., van Lengerich B., Wang J., Robles-Colmenares Y., Kim D.J., Duque J., Lenser M., Earr T.K., Nguyen H., Chau R., Tsogtbaatar B., Ravi R., Skuja L.L., Solanoy H., Rosen H.J., Boeve B.F., Boxer A.L., Heuer H.W., Dennis M.S., Kariolis M.S., Monroe K.M., Przybyla L., Sanchez P.E., Meisner R., Diaz D., Henne K.R., Watts R.J., Henry A.G., Gunasekaran K., Astarita G., Suh J.H., Lewcock J.W., DeVos S.L, & Di Paolo G. (2021). Rescue of a lysosomal storage disorder caused by Grn loss-of-function with a brain penetrant progranulin biologic. Cell, 184(18), 4651-4668.e25.

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