Anti-S6K1 antibody [E343] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunocytochemistry/Immunofluorescence analysis of MCF 7 (Human breast adenocarcinoma epithelial cell) labeling S6K1 with ab32529 at a dilution of 1 : 200, 11.1 ug/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A dilution of 1/1000 (2μg/ml)  was used for the secondary antibodyGoat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained at 1 : 200 dilution, 2.5μg/ml with ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) . Nuclei counterstained with DAPI (blue). Control : 1 : 1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunohistochemical analysis of Human breast cancer tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Overlay histogram showing HeLa cells stained with ab32529 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32529, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Intracellular Flow Cytometry analysis of 293T (Human embryonic kidney epithelial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1 μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) labeling S6K1 with ab32529 at a dilution of 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. ab150077 at 1/1000 was used as the secondary antibody. Nuclei were counterstained with DAPI (blue).

Confocal image showing cytoplamic staining on HeLa cell line.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• IP

Unknown

Immunoprecipitation - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Lane 1 : HEK293T (Human embryonic kidney epithelial cell) whole cell lysate, 10μg
Lane 2 : HEK293T whole cell lysate, 10μg and ab32529, 2μg
Lane 3 : HEK293T cell lysate, 350μg and rabbit IgG (ab172730) , 2μg

Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1 : 500 dilution (4.4 μg/ml). For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

All lanes:

Immunoprecipitation - Anti-S6K1 antibody [E343] (<a href='/en-us/products/primary-antibodies/s6k1-antibody-e343-ab32529'>ab32529</a>)

Predicted band size: 59 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunohistochemical analysis of mouse testis tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Intracellular Flow Cytometry analysis of C6 (Rat glial tumor glial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1 μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunohistochemical analysis of rat brain tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Intracellular Flow Cytometry analysis of Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling with ab32529 (purified) at 1/2200 dilution (1 μg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) labelling with ab32529 at a dilution of 1 : 200, 11.1 μg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1 : 1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained at 1 : 200 dilution, 2.5μg/ml with ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control : 1 : 1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Immunocytochemistry/Immunofluorescence analysis of C6 cells (Rat glial tumor glial cell)  labelling S6K1 with ab32529 at a dilution of 1 : 200, 11.1 µg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1 : 1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained with 1 : 200, 2.5μg/ml with ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control : 1 : 1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

• IP

Unknown

Immunoprecipitation - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

Lane 1 : Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate, 10μg
Lane 2 : Neuro2a whole cell lysate 350μg and ab32529, 2μg
Lane 3 : Neuro2a cell lysate, 350μg and rabbit IgG (ab172730), 2μg

Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1 : 500 dilution (4.4 μg/ml). For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32529).

All lanes:

Immunoprecipitation - Anti-S6K1 antibody [E343] (<a href='/en-us/products/primary-antibodies/s6k1-antibody-e343-ab32529'>ab32529</a>)

Predicted band size: 59 kDa

false

• WB

Lab

Western blot - Anti-S6K1 antibody [E343] - BSA and Azide free (AB203558)

This data was developed using ab32529, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[E343] to S6K1 ab32529 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 59 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in RPS6KB1 knockout U-87 MG cell line (ab326023). To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-S6K1 antibody [E343] (<a href='/en-us/products/primary-antibodies/s6k1-antibody-e343-ab32529'>ab32529</a>) at 1/10000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

Western blot - Human RPS6KB1 knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-rps6kb1-knockout-u-87-mg-cell-line-ab326023'>ab326023</a>) at 20 µg

Lane 2:

RPS6KB1 knockout U-87 MG at 20 µg

Lane 3:

MCF7 at 20 µg

Lane 4:

HEK-293 at 20 µg

Secondary

All lanes:

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

Predicted band size: 59 kDa

Observed band size: 59 kDa

false