Anti ilf3

Manufactured by BD

Anti-ILF3 is a laboratory equipment product designed for the detection and analysis of the interleukin enhancer-binding factor 3 (ILF3) protein. It functions as a tool for researchers to study the expression and localization of ILF3 in various biological samples.

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Lab products found in correlation

Tcs sp2, by Leica (1 mentions) Control igg, by BD (1 mentions) Rabbit anti mouse igg, by Thermo Fisher Scientific (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Anti β actin, by Merck Group (1 mentions) Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions) Alliance ld2 77wl system, by Uvitec (1 mentions)

4 protocols using anti ilf3

Immunofluorescence Staining of ILF3 and DJ-1

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Cells were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed twice in PBS, and treated with glycine 0.1 M in PBS for 5 min. Following 2 more washes in PBS, fixed cells were permeabilized with 0.1% Triton X-100 for 4 min at room temperature and blocked with 0.2% BSA, 1% FBS, and 0.1% Triton in PBS for 5 min. Cells were subsequently incubated 90 min with anti-ILF3 (BD Bioscience) 1:50 in blocking solution, washed in PBS 3 times, and finally stained with AlexaFluor 488– or AlexaFluor 594–labeled anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher Scientific), 1:250 in blocking buffer. Nuclei were visualized with DAPI (1 μg/ml). Anti–DJ-1 1:250 (56 (link)) was used to counterstain cell cytoplasm. Images were captured with a confocal microscope (Leica TCS SP2; Leica Microsystems, Buffalo Grove, IL, USA).

Fasolo F., Patrucco L., Volpe M., Bon C., Peano C., Mignone F., Carninci P., Persichetti F., Santoro C., Zucchelli S., Sblattero D., Sanges R., Cotella D, & Gustincich S. (2019). The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs. The FASEB Journal, 33(12), 13572-13589.

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Profiling ILF3 RNA Interactome

Cited 1 time

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ILF3 RIP was performed following standard methods (Feng et al., 2014 (link)). 4.5x10⁷ MCF10A-DCIS cells were lysed in RIP buffer (150mM KCl, 25mM Tris pH 7.4, 5mM EDTA, 0.5% NP40, protease and RNAse inhibitors), precleared with streptavidin beads, and incubated overnight with 10ug either control IgG or anti-ILF3 (BD Biosciences cat #612154). Streptavidin beads were added for an additional 2hr incubation. Beads were washed with RIP buffer, incubated in TRIzol, and RNA was purified via chloroform extraction.

DeVaux R.S., Ropri A.S., Grimm S.L., Hall P.A., Herrera E.O., Chittur S.V., Smith W.P., Coarfa C., Behbod F, & Herschkowitz J.I. (2020). Long noncoding RNA BHLHE40-AS1 promotes early breast cancer progression through modulating IL6 / STAT3 signaling. Journal of cellular biochemistry, 121(7), 3465-3478.

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Antibody Immunoprecipitation Optimization

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Anti-hnRNP-C (4F4), anti-HuR (N-16), and anti-PTBPl (N-20) were purchased from Santa Cruz Biotechnology. Anti-ILF3 (cat# 612155) was purchased from BD Biosciences. Anti-DDX5 was purchased from Abeam (cat# ab2l696). Antibodies were preconjugated to Protein G Dynabeads (Thermo Fisher Scientific, cat # 10004D) overnight in PBS. Rabbit anti-mouse IgG, purchased from Thermo Fisher Scientific (cat# 31188), was added to overnight preconjugations of primary mouse antibodies at a 1:1 ratio. For immunoblots, antibodies were used at 1:1,000. For immunoprecipitations, 1 μg of antibody was used per 50 μg of lysate.

Zarnegar B.J., Flynn R.A., Shen Y., Do B.T., Chang H.Y, & Khavari P.A. (2016). irCLIP platform for efficient characterization of protein—RNA interactions. Nature methods, 13(6), 489-492.

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Western Blot and RNA-IP Analysis

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For Western blot analysis, cell pellets were directly dissolved in Laemmli sample buffer. For RNA-IP experiments, ILF3 immunoprecipitation efficiency was monitored by loading the whole fraction of proteins recovered from the organic phase after Trizol extraction, following resuspension in Laemmli sample buffer. All lysates were briefly sonicated, boiled, and loaded on 10% polyacrylamide gels. Immunoblotting was performed with the following primary antibodies: anti-ILF3 (612154; BD Biosciences), 1:500 overnight, and anti–β-actin (A5441; MilliporeSigma), 1:2000. Signals were revealed after incubation with HRP secondary antibodies (Agilent Technologies, Santa Clara, CA, USA) 1:1000 for 1 h at room temperature, in combination with ECL (GE Healthcare). Image detection was performed with Alliance LD2-77WL system (Uvitec, Cambridge, United Kingdom). Image quantification was done using ImageJ software.

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