Anti-14-3-3 alpha + beta antibody [Y62] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-14-3-3 alpha + beta antibody [Y62] - BSA and Azide free (AB247270)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling 14-3-3 alpha + beta with purified ab32560 at 1/100 dilution (10 μg/mL). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 μg/mL) was used as the secondary antibody only control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32560).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-14-3-3 alpha + beta antibody [Y62] - BSA and Azide free (AB247270)

This data was developed using ab32560, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of Hela (human cervical adenocarcinoma epithelial cell) cells labelling 14-3-3 alpha + beta with ab32560 at 1/1000 dilution (0.1ug) (Red) compared with a Secondary Antibody - Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150081) and Isotype control - Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-14-3-3 alpha + beta antibody [Y62] - BSA and Azide free (AB247270)

Flow cytometric analysis of NIH/3T3 (mouse embryonic fibroblast) cells labelling 14-3-3 alpha + beta with ab32560 at 1/1000 dilution (0.1ug) (Red) compared with a Secondary Antibody - Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150081) and Isotype control - Rabbit monoclonal IgG (ab172730) (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

• IP

Lab

Immunoprecipitation - Anti-14-3-3 alpha + beta antibody [Y62] - BSA and Azide free (AB247270)

This data was developed using ab32560, the same antibody clone in a different buffer formulation.

14-3-3 alpha + beta was immunoprecipitated from 10ug NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab32560 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab32560 at 1/1000 dilution.

VeriBlot for IP secondary antibody(HRP) (ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-14-3-3 alpha + beta antibody [Y62] (<a href='/en-us/products/primary-antibodies/14-3-3-alpha-beta-antibody-y62-ab32560'>ab32560</a>) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/14-3-3-alpha-beta-antibody-y62-ab32560'>ab32560</a> at 1/30 in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/14-3-3-alpha-beta-antibody-y62-ab32560'>ab32560</a> in NIH/3T3 whole cell lysate at 10 µg

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

• WB

Lab

Western blot - Anti-14-3-3 alpha + beta antibody [Y62] - BSA and Azide free (AB247270)

This data was developed using ab32560, the same antibody clone in a different buffer formulation.

In Western blot, Anti-6X His tag® antibody [AD1.1.10] (ab15149) total protein control and secondary antibody used was Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/20000 dilution.

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

ab32560 does not react with other 14-3-3 protein families.

All lanes:

Western blot - Anti-14-3-3 alpha + beta antibody [Y62] (<a href='/en-us/products/primary-antibodies/14-3-3-alpha-beta-antibody-y62-ab32560'>ab32560</a>) at 1/1000 dilution

Lane 1:

Human 14-3-3 beta (YWHAB) Recombinant Protein with a his tag

Lane 2:

Human 14-3 3 gamma (YWHAG) Recombinant Protein with a his tag

Lane 3:

Human 14-3-3 epsilon (YWHAE) Recombinant Protein with a his tag

Lane 4:

Human 14-3-3 zeta (YWHAZ) Recombinant Protein with a his tag

Lane 5:

Human 14 3 3 eta (YWHAH) Recombinant Protein with a his tag

Lane 6:

Human 14-3-3 theta (YWHAQ) Recombinant Protein with a his tag

Lane 7:

Human 14-3-3 sigma (SFN) Recombinant Protein with a his tag

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: 30 kDa

false

Exposure time: 42s