Anti-CPEB4 antibody [EPR27416-141]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U266B1 (human multiple myeloma B lymphocyte) cells labelling CPEB4 with ab315332 at 1/50 (10.32 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 cytoplasmic staining in U266B1 cells. Low expression : A-204. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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-CPEB4 antibody [EPR27416-141] (AB315332)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A-204 (human muscle rhabdomyosarcoma cell, Left) / U266B1 (human multiple myeloma B lymphocyte, Right) cells labelling CPEB4 with ab315332 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).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Low expression : A-204.

• IP

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Immunoprecipitation - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

CPEB4 was immunoprecipitated from 0.35 mg U266B1 (human multiple myeloma B lymphocyte) whole cell lysate with ab315332 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315332 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-CPEB4 antibody [EPR27416-141] (ab315332) at 1/1000 dilution

Lane 1:

U266B1 (human multiple myeloma B lymphocyte) whole cell lysate

Lane 2:

ab315332 at 1/30 IP in U266B1 (human multiple myeloma B lymphocyte) whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab315332 in U266B1 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 119s

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse hippocampus (fresh frozen) tissue labeling CPEB4 with ab315332 at 1/100 (5.16 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Confocal image showing positive staining on mouse hippocampus. The nuclear counterstain was DAPI (Blue). The section was incubated with ab315332 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labelling CPEB4 with ab315332 at 1/50 (10.32 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 cytoplasmic staining in Neuro-2a cells and showing weak staining in F9 cells. Low expression : F9. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse kidney (fresh frozen) tissue labeling CPEB4 with ab315332 at 1/100 (5.16 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Low expression : confocal image showing no staining on mouse kidney (PMID : 17024188). The nuclear counterstain was DAPI (Blue). The section was incubated with ab315332 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse skeletal muscle (fresh frozen) tissue labeling CPEB4 with ab315332 at 1/100 (5.16 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Low expression : confocal image showing no staining on mouse skeletal muscle (PMID : 17024188). The nuclear counterstain was DAPI (Blue). The section was incubated with ab315332 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-Fr

Supplier Data

Immunohistochemistry (Frozen sections) - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen Mouse hippocampus (fresh frozen) tissue labeling CPEB4 with ab315332 at 1/100 (5.16 ug/ml) dilution followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution (Green).

Panel A : merged staining of anti-CPEB4 (ab315332, green), anti-NeuN (ab190565, grey) and anti-GFAP (ab201732, red) on mouse hippocampus. Panel B : anti-CPEB4 stained on mouse hippocampus. Panel C : anti-NeuN stained in neurons of mouse hippocampus. Panel D : anti-GFAP stained in astrocytes of mouse hippocampus. The nuclear counterstain was DAPI (Blue). The section was incubated with ab315332 for 60 mins at room temperature. The section was then mounted using Fluoromount®. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). The nuclear counterstain was DAPI (Blue).

Secondary antibody control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• WB

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Western blot - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Low expression : A-204, A-673, F9.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

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

All lanes:

Western blot - Anti-CPEB4 antibody [EPR27416-141] (ab315332) at 1/1000 dilution

Lane 1:

U266B1 (human mtiple myeloma B lymphocyte) whole cell lysate at 50 µg

Lane 2:

A-204 (rat muscle rhabdomyosarcoma) whole cell lysate at 50 µg

Lane 3:

A-673 (human muscle ewis sarcoma) whole cell lysate at 50 µg

Lane 4:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 50 µg

Lane 5:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 90 kDa,36 kDa

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Low expression : kidney (PMID : 17024188).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

Exposure time : Lane 1-7 : 10 seconds, Lane 8-9 : 48 seconds.

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

All lanes:

Western blot - Anti-CPEB4 antibody [EPR27416-141] (ab315332) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 50 µg

Lane 2:

Mouse cerebellum tissue lysate at 50 µg

Lane 3:

Mouse kidney tissue lysate at 50 µg

Lane 4:

Rat brain tissue lysate at 50 µg

Lane 6:

Rat hippocampus tissue lysate at 50 µg

Lane 7:

Rat kidney tissue lysate at 50 µg

Lane 8:

Human brain tissue lysate at 50 µg

Lane 9:

Human cerebellum tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 90 kDa,36 kDa

false

• WB

Lab

Western blot - Anti-CPEB4 antibody [EPR27416-141] (AB315332)

Western blot : Anti-CPEB4 antibody [EPR27416-141] (ab315332) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab315332 was shown to bind specifically to CPEB4. A band was observed at 80 kDa in wild-type A549 cell lysates with no signal observed at this size in CPEB4 knockout cell line. To generate this image, wild-type and CPEB4 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CPEB4 antibody [EPR27416-141] (ab315332) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

CPEB4 knockout A549 cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 80 kDa

false