Anti-CUG-BP1 antibody [3B1]

• ICC

Lab

Immunocytochemistry - Anti-CUG-BP1 antibody [3B1] (AB9549)

ab9549 staining CUG-BP1 in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 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 ab9549 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 100% methanol (5 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

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CUG-BP1 antibody [3B1] (AB9549)

IHC image of CUG-BP1 staining in human normal kidney formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ 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 ab9549, 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.

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

• WB

Lab

Western blot - Anti-CUG-BP1 antibody [3B1] (AB9549)

Lanes 1-4 : Merged signal (red and green). Green - ab9549 observed at 52 kDa. Red - loading control ab181602 observed at 36 kDa.

ab9549 Anti-CUG-BP1 antibody [3B1] was shown to specifically react with CUG-BP1 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266086 (knockout cell lysate ab257390) was used. Wild-type and CUG-BP1 knockout samples were subjected to SDS-PAGE. ab9549 and Anti-GAPDH antibody[EPR16891] - Loading Control (ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-CUG-BP1 antibody [3B1] (ab9549) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

CELF1 knockout HEK293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Human brain tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-CUG-BP1 antibody [3B1] (AB9549)

All lanes:

Western blot - Anti-CUG-BP1 antibody [3B1] (ab9549) at 1 µg/mL

Lane 1:

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

Lane 2:

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

Lane 3:

Human kidney tissue lysate - total protein (<a href='/en-us/products/unavailable/human-kidney-tissue-lysate-total-protein-ab30203'>ab30203</a>) at 10 µg

Secondary

All lanes:

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

Predicted band size: 52 kDa

Observed band size: 54 kDa

true

Exposure time: 20min