Anti bim antibody

Manufactured by Cell Signaling Technology

Sourced in United States

The Anti-Bim antibody is a laboratory reagent used for the detection and analysis of the Bim protein, a member of the Bcl-2 family of proteins involved in the regulation of apoptosis (programmed cell death). This antibody can be used in various immunoassay techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and localization of Bim in biological samples.

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Anti-Bim (89 citations) BIM antibody (8 citations) Rabbit anti-Bim antibody (4 citations)

5 protocols using anti bim antibody

Antibody Staining for Flow Cytometry

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Anti-TRIM33 antibody (A301-060A; Bethyl Laboratories, Montgomery, TX), Anti-PU.1 antibody (#2266; Cell Signaling, Beverly, MA or sc-352; Santa Cruz, Dallas, TX), Anti-Bim antibody (#2819; Cell Signaling), Anti-H3K27ac antibody (ab4729; Abcam, Cambridge, MA), Anti-H3K4me3 antibody (07-473; Millipore), Anti-ß-actin HRP antibody (#A3854; Sigma, Ronkonkoma, NY), APC anti-mouse B220 (#103212; BioLegend, San Diego, CA), APC anti-mouse CD-19, APC anti-mouse Mac-1/Cd11b (#101211; BioLegend), APC anti-mouse Ly-6G/Gr-1 (#17-5931; eBioscience, San Diego, CA), APC anti-mouse TER-119 (#116212; BioLegend), APC anti-mouse CD-3 (#100209; BioLegend).

Wang E., Kawaoka S., Roe J.S., Shi J., Hohmann A.F., Xu Y., Bhagwat A.S., Suzuki Y., Kinney J.B, & Vakoc C.R. (2015). The transcriptional cofactor TRIM33 prevents apoptosis in B lymphoblastic leukemia by deactivating a single enhancer. eLife, 4, e06377.

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Protein Expression Analysis by Western Blot

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Total protein was extracted with RIPA buffer and quantified with bicinchoninic acid (BCA) protein quantitative assay (Thermo, USA). The samples were separated by 10% SDS-PAGE and transferred into PVDF membranes (Merck Millipore, USA). The membranes were blocked in 3% Bovine Serum Albumin (BSA) with PBS. Then, the membranes were incubated with corresponding antibodies: anti-ADSL antibody (Abcam, United Kingdom), anti-β-actin antibody (Cell Signaling Technology, USA), anti-Rb antibody (Cell Signaling Technology), anti-p21 antibody (Cell Signaling Technology), anti-CDK4 antibody (Cell Signaling Technology), anti-CDC2 antibody (Cell Signaling Technology), anti-Bcl2 antibody (Abcam), anti-Bax antibody (Abcam), anti-p27 antibody (Cell Signaling Technology), anti-Bid antibody (Cell Signaling Technology), anti-Bim antibody (Cell Signaling Technology). Appropriate second antibodies were applied in the next incubation. Lastly, the enhanced chemiluminescence (ECL) detection system (ImageQuant LAS 500, USA) was used to detect the membranes following the manufacturer’s protocol.

Liao J., Song Q., Li J., Du K., Chen Y., Zou C, & Mo Z. (2021). Carcinogenic effect of adenylosuccinate lyase (ADSL) in prostate cancer development and progression through the cell cycle pathway. Cancer Cell International, 21, 467.

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BIM Protein Immunoprecipitation in Cell Lysates

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IP was performed using 400-μg whole-cell protein lysates isolated in 1ml RIPA lysis buffer. To reduce unspecific bead binding, lysates were pre-incubated with 50μl protein G sepharose beads (GE Healthcare, USA) for 2h followed by overnight incubation with 5μl anti-BIM antibody (Cell Signaling, USA) at 4°C. protein G sepharose beads were added for a 2-h incubation at 4°C. Beads were sedimented and washed with PBS, and proteins were denatured and dissolved in 30μl Laemmli buffer (Bio-Rad, Germany) at 95°C for 5 min. Fifteen microliters of the samples was immunoblotted as described above.

Müller D., Mazzeo P., Koch R., Bösherz M.S., Welter S., von Hammerstein-Equord A., Hinterthaner M., Cordes L., Belharazem D., Marx A., Ströbel P, & Küffer S. (2021). Functional apoptosis profiling identifies MCL-1 and BCL-xL as prognostic markers and therapeutic targets in advanced thymomas and thymic carcinomas. BMC Medicine, 19, 300.

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Immunoprecipitation of Bim and Noxa

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Cells treated with different drugs were lysed with NP-40 lysis buffer [50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 0.5% NP-40, 50 mmol/L NaF, 0.2 mmol/L Na₃VO₄, and 1 mmol/L DTT] for 120 min. Total protein (400 µg) was first precleared with 20 µl protein A/G plus-agarose (Santa Cruz Bio-technology) and then subjected to IP with anti-Bim antibody (Cell Signaling Technology, Inc.) or anti-Noxa antibody (Cell Signaling Technology, Inc.) at 4 °C for overnight. Twenty microliter of protein A/G plus-agarose beads was added and incubated for 2 h to pull down protein-antibody complexes. The beads were spun, washed four times with NP-40 lysis buffer, resuspended in 2X SDS sample buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol], and heated at 98 °C for 5 min for analysis by SDS-polyacrylamide gel electrophoresis and Western blotting.

Zhang J., Wang Y., Yin C., Gong P., Zhang Z., Zhao L., Waxman S, & Jing Y. (2022). Artesunate improves venetoclax plus cytarabine AML cell targeting by regulating the Noxa/Bim/Mcl-1/p-Chk1 axis. Cell Death & Disease, 13(4), 379.

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Enrichment and Protein Analysis of CD69+ T Cells

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CD69⁺ and CD69⁻ T cells were enriched from thymocytes using biotinylated anti-CD69 antibody (BioLegend) and MojoSort Streptavidin Nanobeads (BioLegend). CD69^- T cells were further purified by depleting TCRβ⁺ cells using biotinylated anti-TCRβ antibody (BioLegend) and MojoSort Streptavidin Nanobeads (BioLegend). Expression of Bim and ACTB proteins in each subset was detected with anti-Bim antibody (1:1000, #2819, Cell Signaling, MA, USA) and anti-ACTB antibody (1:10,000, NB600-532, Novus Biologicals, CO, USA). As the secondary antibody, anti-rabbit IgG (1:15,000, 711-035-152, Jackson ImmumoResearch, PA, USA) was used. Signals were visualized with ECL plus Western Blotting Detection Reagents (GE Healthcare) and analyzed by the CCD digital imaging system LAS-4000 Luminescent Image Analyzer (GE Healthcare). Whole proteins were stained with SYPRO Ruby Protein Gel Stain (S12000, Thermo Fisher Scientific) and detected by the CCD digital imaging system LAS-4000 Luminescent Image Analyzer (GE Healthcare). Uncropped scans are available in the Source data file.

Hojo M.A., Masuda K., Hojo H., Nagahata Y., Yasuda K., Ohara D., Takeuchi Y., Hirota K., Suzuki Y., Kawamoto H, & Kawaoka S. (2019). Identification of a genomic enhancer that enforces proper apoptosis induction in thymic negative selection. Nature Communications, 10, 2603.

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