Anti-Coilin antibody [IH10]

• ICC

Lab

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 ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution (shown in green) and 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.

• IHC-P

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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 (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

• ICC

AbReview23398****

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.

This image is part of an abreview submitted by Kirk McManus, Univ. of Manitoba/Cancer Care MICB, Canada

• IHC-P

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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.

• Flow Cyt (Intra)

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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 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

• IP

Unknown

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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• WB

Project

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 (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-hrp-preadsorbed-ab97040'>ab97040</a>) at 1/5000 dilution

Predicted band size: 62 kDa

Observed band size: 28 kDa,75 kDa

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Exposure time: 20min

• WB

Lab

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

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Exposure time: 12min

• WB

Unknown

Western blot - Anti-Coilin antibody [IH10] (AB87913)

Lanes 1-3 : Merged signal (red and green). Green - ab87913 observed at 75 kDa. Red - 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 ab261757 (knockout cell lysate ab257251) was used. Wild-type and Coilin knockout samples were subjected to SDS-PAGE. ab87913 and 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 (ab216772) and 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 (<a href='/en-us/products/cell-lines/human-coil-coilin-knockout-hela-cell-line-ab261757'>ab261757</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Predicted band size: 62 kDa

Observed band size: 75 kDa

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