Anti-Coilin antibody [IH10]

9 product images

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  Immunocytochemistry - Anti-Coilin antibody [IH10] (ab87913)

  ab87913 staining Coilin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab87913 at 1µg/ml and Anti-beta Tubulin antibody - Loading Control ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

  Also suitable in cells fixed with 4% paraformaldehyde (10 min).

  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

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  This image is part of a customer review submitted by Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

  Immunocytochemistry - Anti-Coilin antibody [IH10] (ab87913)

  ab87913(1/2000) staining Coilin in assynchronous HeLa cells (green). Cells were fixed in methanol (this image) or paraformaldehyde (see abreview image), permeabilized with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). For further experimental details please refer to Abreview.

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  Flow Cytometry (Intracellular) - Anti-Coilin antibody [IH10] (ab87913)

  Overlay histogram showing HeLa cells stained with ab87913 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab87913, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (Mouse IgG2b [PLPV219] - Isotype Control ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.
  
  

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  Western blot - Anti-Coilin antibody [IH10] (ab87913)

  Blocking buffer: 3% Milk
  

  Gel type: MOPS

  The band observed in the CRISPR-Cas9 edited lysate lane below 75 kDa is likely to represent a truncated form of COIL. This has not been investigated further and the functional properties of the gene product have not been determined.

  All lanes: Western blot - Anti-Coilin antibody [IH10] (ab87913) at 1/500 dilution

  Lane 1: Jurkat whole cell at 20 µg

  Lane 2: Wild-type HeLa cell lysate at 20 µg

  Lane 3: Empty

  Lane 4: COIL knockout HeLa cell lysate at 20 µg

  Secondary

  All lanes: Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/5000 dilution

  Predicted band size: 62 kDa

  Observed band size: 75 kDa

  Exposure time: 12min

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  Western blot - Anti-Coilin antibody [IH10] (ab87913)

  Lanes 1-3: Merged signal (red and green). Green - ab87913 observed at 75 kDa. Red - loading control, Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 observed at 37 kDa.
  

  ab87913 Anti-Coilin antibody [IH10] was shown to specifically react with Coilin in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human COIL (Coilin) knockout HeLa cell line ab261757 (knockout cell lysate Human COIL (Coilin) knockout HeLa cell lysate ab257251) was used. Wild-type and Coilin knockout samples were subjected to SDS-PAGE. ab87913 and Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Coilin antibody [IH10] (ab87913) at 1/500 dilution

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: COIL knockout HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human COIL (Coilin) knockout HeLa cell line (Human COIL (Coilin) knockout HeLa cell line ab261757)

  Lane 3: Jurkat cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 62 kDa

  Observed band size: 75 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Coilin antibody [IH10] (ab87913)

  IHC image of Coilin staining in human testis formalin fixed paraffin embedded tissue section*. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab87913, 3μg/ml overnight at +4°C. An HRP-conjugated secondary (Goat Anti-Mouse IgG2b heavy chain (HRP) ab97250, 1/500 dilution) was used for 1hr at room temperature. The section was counterstained with haematoxylin and mounted with DPX.

  The inset negative control image is taken from an identical assay without primary antibody.

  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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  Immunoprecipitation - Anti-Coilin antibody [IH10] (ab87913)

  Coilin was immunoprecipitated using 0.5mg HepG2 whole cell extract, 5µg of Mouse monoclonal to Coilin and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
  The antibody was incubated under agitation with Protein G beads for 10min, HepG2 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
  Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab87913.
  Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
  Band: 75kDa: Coilin

  All lanes: Immunoprecipitation - Anti-Coilin antibody [IH10] (ab87913)

  Predicted band size: 62 kDa

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  Western blot - Anti-Coilin antibody [IH10] (ab87913)

  All lanes: Western blot - Anti-Coilin antibody [IH10] (ab87913) at 5 µg/mL

  Lane 1: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 2: HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg

  Lane 3: Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate at 10 µg

  Lane 4: HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 5: HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

  Lane 6: MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 7: Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 8: SHSY-5Y (Human neuroblastoma cell line) Whole Cell Lysate at 10 µg

  Secondary

  All lanes: Western blot - Goat Anti-Mouse IgG H&L (HRP) preadsorbed (Goat Anti-Mouse IgG H&L (HRP) preadsorbed ab97040) at 1/5000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 62 kDa

  Observed band size: 28 kDa, 75 kDa

  Exposure time: 20min

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Coilin antibody [IH10] (ab87913)

  IHC image of Coilin staining in Human normal testis formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab87913, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
  

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.