4336 apc 050

Manufactured by Cell Signaling Technology

Sourced in United States

4336-APC-050 is a fluorescently-labeled secondary antibody product from Cell Signaling Technology. It is conjugated to the fluorescent dye Allophycocyanin (APC).

Automatically generated - may contain errors

Lab products found in correlation

Ab1790, by Abcam (1 mentions) Ab3749, by Abcam (1 mentions) Ab63801, by Abcam (1 mentions) Ab16048, by Abcam (1 mentions) Universal magnetic co ip kit, by Active Motif (1 mentions) Parp 1, by BD (1 mentions) Edta free protease inhibitor mixture, by Roche (1 mentions) β actin, by Cell Signaling Technology (1 mentions)

2 protocols using 4336 apc 050

Fractionation and Immunoprecipitation of Nuclear Protein Complexes

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To resolve nuclear multiprotein complexes, nuclear extracts from S1 cells (23 (link)) were loaded onto a 10–40% sucrose gradient and ultracentrifuged for 40 h at 4°C and 214 000 g. Fractions of equal volumes were precipitated with trichloroacetic acid and analyzed with the pellet (insoluble fraction) by western blot. In immunoprecipitation (IP) experiments, nuclear extracts (1 mg) were incubated with antibodies overnight at 4°C and further processed using the Universal Magnetic Co-IP kit (Active Motif) according to the manufacturer's instructions. Antibodies used for immunoblotting were: 53BP1 (Abcam, Ab36823, 1 μg/ml), BRCA1 (Calbiochem, MS110, 5 μg/ml), BRG1 (Milipore, 07–478, 1:10000), DNA-PKcs (Abcam, clone 18–2, 2 μg/ml), γH2AX (Ser139; Millipore, clone JBW301, 1 μg/ml), Histone H2B (Abcam, Ab1790, 0.1 μg/ml), lamin B (Abcam, Ab16048, 60 ng/ml), NuMA (B1C11, 1:2, a gift from Dr Jeffrey Nickerson, UMass, Worcester, USA), PAR (Trevigen, 4336-APC-050, 1:1000), phospho-NuMA (Cell Signaling, 1:1000), SNF2h (Abcam, Ab3749, 1 μg/ml), RAD51 (Abcam, Ab63801, 1:1000), tubulin (Abcam, Ab3194, 1 μg/ml) and Williams Syndrome Transcription Factor (WSTF; Cell Signaling, 0.3 μg/ml). For IP: NuMA (Oncogene, clone Ab-2 or Bethyl Laboratories) and SNF2h (Abcam, clone 3.25(2)).

Vidi P.A., Liu J., Salles D., Jayaraman S., Dorfman G., Gray M., Abad P., Moghe P.V., Irudayaraj J.M., Wiesmüller L, & Lelièvre S.A. (2014). NuMA promotes homologous recombination repair by regulating the accumulation of the ISWI ATPase SNF2h at DNA breaks. Nucleic Acids Research, 42(10), 6365-6379.

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SDS-PAGE Protein Extraction and Analysis

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SDS-PAGE and transfer were performed according to laboratory protocol with slight modification. In brief, cultured cells were lysed in RIPA buffer containing 1% Triton, 0.5% Na-deoxycholate, 0.1% SDS, 50 mM NaF, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, and EDTA-free protease inhibitor mixture (Roche Diagnostics) in PBS (pH7.4). Protein extracts were separated by 4–12% SDS-PAGE. Proteins were transferred at constant voltage of 80 V for 150 min at 4°C from the SDS to PVDF membranes. Membrane were then blocked with 5% non-fat milk and incubated with primary antibodies overnight at 4°C. Antibodies used were anti-PAR (Trevigen, 4336-APC-050), Actin (Cell Signaling, 5125), PARP-1 (BD, 611039), β-Actin (Cell Signaling Technology, #5125S). After washes with TBST (TBS with 0.1% Tween-20), membranes were incubated with HRP-conjugated secondary antibodies for 1 hr at RT. Immunoreactive bands were visualized by the enhanced chemiluminescent substrate (ECL, Pierce) on X-ray film and quantified using the image software TINA.

Xu J.C., Fan J., Wang X., Eacker S.M., Kam T.I., Chen L., Yin X., Zhu J., Chi Z., Jiang H., Chen R., Dawson T.M, & Dawson V.L. (2016). Cultured networks of excitatory projection neurons and inhibitory interneurons for studying human cortical neurotoxicity. Science translational medicine, 8(333), 333ra48.

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