Anti-UNC5B antibody [EPR26173-186]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-UNC5B antibody [EPR26173-186] (AB313565)

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling UNC5B with ab313565 at 1/50 (10.56 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing membranous staining in Neuro-2a cells.Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-UNC5B antibody [EPR26173-186] (AB313565)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling UNC5B with ab313565 at 1/50 dilution (1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• WB

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Western blot - Anti-UNC5B antibody [EPR26173-186] (AB313565)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Negative control : Daudi, Raji(PMID : 12655055), HL-60 (PMID : 12655055, PMID : 25171009).

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution.

All lanes:

Western blot - Anti-UNC5B antibody [EPR26173-186] (ab313565) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 4:

HL-60 (human Acute Promyelocytic Leukemia promyeloblast) whole cell lysate at 20 µg

Lane 5:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 50 kDa,65 kDa,130 kDa

false

Exposure time: 26s

• WB

Lab

Western blot - Anti-UNC5B antibody [EPR26173-186] (AB313565)

Western blot : Anti-UNC5B antibody [EPR26173-186] ab313565 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 120, 50 kDa in Wild-type U-87 MG cell lysates with a truncated product observed at 100 kDa in CRISPR-Cas9 Edited cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^™ 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-UNC5B antibody [EPR26173-186] (ab313565) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG (UNBOILED) whole cell lysate at 20 µg

Lane 2:

UNC5B knockout U-87 MG (UNBOILED) whole cell lysate at 20 µg

Lane 3:

SH-SY5Y (UNBOILED) whole cell lysate at 20 µg

Lane 4:

U-2 OS (UNBOILED) whole cell lysate at 20 µg

Lane 5:

Daudi (UNBOILED) whole cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 104 kDa

Observed band size: 120 kDa,50 kDa

false

• WB

Supplier Data

Western blot - Anti-UNC5B antibody [EPR26173-186] (AB313565)

Blocking and diluting buffer and concentration : 5% NFDM/TBST In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-UNC5B antibody [EPR26173-186] (ab313565) at 1/1000 dilution

Lane 1:

Mouse spinal cord tissue lysate at 20 µg

Lane 2:

Mouse brain tissue lysate at 20 µg

Lane 3:

bEnd.3 (mouse brain endothelial cell) while cell lysate at 20 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 5:

Rat brain tissue lysate at 20 µg

Lane 6:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 50 kDa,65 kDa,130 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-UNC5B antibody [EPR26173-186] (AB313565)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody has no cross reaction with mouse UNC5A, UNC5C and UNC5D. In Western blot, anti-His antibody (ab213204) staining at 1/5000 dilution. Exposure time : 3 seconds

All lanes:

Western blot - Anti-UNC5B antibody [EPR26173-186] (ab313565) at 1/1000 dilution

Lane 1:

293T transfected with a mouse UNC5A expression vector containi a His tag whole cell lysate at 40 µg

Lane 2:

293T transfected with a mouse UNC5B vector containi a His tag whole cell lysate at 2 µg

Lane 3:

293T transfected with a mouse UNC5C vector containi a His tag whole cell lysate at 10 µg

Lane 4:

293T transfected with a mouse UNC5D vector containi a His tag whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 130 kDa

false

Exposure time: 3s