Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized 293T (human embryonic kidney epithelial cell) cells labelling 14-3-3 zeta with ab317438 at 1/50 (9.86 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 293T cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). 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 (Magenta). 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)

Supplier Data

Flow Cytometry (Intracellular) - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (human embryonic kidney epithelial cell) cells labelling 14-3-3 zeta with ab317438 at 1/500 dilution (0.1ug)/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.

• IP

Supplier Data

Immunoprecipitation - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

14-3-3 zeta was immunoprecipitated from 0.35 mg 293T (human embryonic kidney epithelial cell) whole cell lysate with ab317438 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317438 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : ab317438 IP in 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317438 in 293T whole cell lysate

All lanes:

Immunoprecipitation - Anti-14-3-3 zeta antibody [EPR27044-19] (<a href='/en-us/products/primary-antibodies/14-3-3-zeta-antibody-epr27044-19-ab317438'>ab317438</a>) at 1/30 dilution

All lanes:

293T (human embryonic kidney epithelial cell) 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: 6s

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 100% methanol-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labelling 14-3-3 zeta with ab317438 at 1/50 (9.86 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 RAW 264.7 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). 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 (Magenta). 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.

• WB

Supplier Data

Western blot - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

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

All lanes:

Western blot - Anti-14-3-3 zeta antibody [EPR27044-19] (<a href='/en-us/products/primary-antibodies/14-3-3-zeta-antibody-epr27044-19-ab317438'>ab317438</a>) at 1/1000 dilution

Lane 1:

293T (human embryonic kidney epithelial cell) transfected with scrambled siRNA control whole cell lysate at 5 µg with NFDM/TBST

Lane 2:

293T transfected with siRNA specifically targeting 14-3-3 zeta whole cell lysate at 5 µg with NFDM/TBST

Lane 3:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 4:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 5:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 6:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg with NFDM/TBST

Lane 7:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg with NFDM/TBST

Lane 8:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg with NFDM/TBST

Lane 9:

C6 (rat glial tumor glial cell) whole cell lysate at 20 µg with NFDM/TBST

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 1/100000 dilution

Observed band size: 27 kDa,36 kDa

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

Lanes 1-2 are applied with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 and lanes 3-4 are applied with Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000.

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

Exposure time : Lanes 1-2 : 8 seconds; lanes 3-4 : 1 second

All lanes:

Western blot - Anti-14-3-3 zeta antibody [EPR27044-19] (<a href='/en-us/products/primary-antibodies/14-3-3-zeta-antibody-epr27044-19-ab317438'>ab317438</a>) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Lane 3:

Human lung cancer tissue lysate at 20 µg

Lane 4:

Human cerebellum tissue lysate at 20 µg

Secondary

Lanes 1 - 2:

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 1/100000 dilution

Lanes 3 - 4:

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

Observed band size: 27 kDa,36 kDa

false

• WB

Supplier Data

Western blot - Anti-14-3-3 zeta antibody [EPR27044-19] - BSA and Azide free (AB317439)

This data was developed using ab317438, the same antibody clone in a different buffer formulation.

This antibody does not cross-react with human 14-3-3 epsilon, 14-3-3 sigma, 14-3-3 eta, 14-3-3 gamma, 14-3-3 tau and 14-3-3 beta.

In Western blot, Anti-6X His tag® antibody [EPR20547] - ChIP Grade (ab213204) staining at 1/5000 dilution.

All lanes:

Western blot - Anti-14-3-3 zeta antibody [EPR27044-19] (<a href='/en-us/products/primary-antibodies/14-3-3-zeta-antibody-epr27044-19-ab317438'>ab317438</a>) at 1/1000 dilution

Lane 1:

His-tagged full-length human 14-3-3 zeta / YWHAZ recombinant protein at 10 ng with NFDM/TBST

Lane 2:

His-tagged full-length human 14-3-3 epsilon / YWHAE recombinant protein at 10 ng with NFDM/TBST

Lane 3:

His-tagged full-length human 14-3-3 sigma / YWHAS recombinant protein at 10 ng with NFDM/TBST

Lane 4:

His-tagged full-length human 14-3-3 eta / YWHAH recombinant protein at 10 ng with NFDM/TBST

Lane 5:

His-tagged full-length human 14-3-3 gamma / YWHAG recombinant protein at 10 ng with NFDM/TBST

Lane 6:

His-tagged full-length human 14-3-3 tau / YWHAQ recombinant protein at 10 ng with NFDM/TBST

Lane 7:

His-tagged full-length human 14-3-3 beta / YWHAB recombinant protein at 10 ng with NFDM/TBST

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 1/100000 dilution

Observed band size: 27 kDa

false

Exposure time: 81s