Anti-PYK2 antibody [YE353]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (AB32571)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling PYK2 with purified ab32571 at 1/60. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

Control : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (AB32571)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human cerebral cortex tissue labelling PYK2 with purified ab32571 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (AB32571)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal human brain tissue labelling PYK2 with unpurified ab32571 at a 1/250 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (AB32571)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebral cortex tissue labelling PYK2 with purified ab32571 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PYK2 antibody [YE353] (AB32571)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat cerebral cortex tissue labelling PYK2 with purified ab32571 at 1/300. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PYK2 antibody [YE353] (AB32571)

Immunocytochemistry/Immunofluorescence analysis of PC12 cells labelling PYK2 with unpurified ab32571 at a 1/100 dilution.

• WB

Lab

Western blot - Anti-PYK2 antibody [YE353] (AB32571)

Western blot : Rabbit Monoclonal[YE353] to PYK2 ab32571 staining at 1/2000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta.

A band was observed at 120 kDa in Wild-type HCT 116 cell lysates with no signal observed at this size in PTK2B knockout HCT 116 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 & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-PYK2 antibody [YE353] (ab32571) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 at 20 µg

Lane 2:

PTK2B knockout HCT 116 at 20 µg

Lane 3:

Wild-type A549 at 20 µg

Lane 4:

PTK2B knockout A549 at 20 µg

Secondary

All lanes:

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

Predicted band size: 116 kDa

Observed band size: 120 kDa

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• WB

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Western blot - Anti-PYK2 antibody [YE353] (AB32571)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : PYK2 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : Jurkat whole cell lysate (20 μg)
Lane 4 : Hu brain whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab32571 observed at 125 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32571 was shown to specifically recognize PYK2 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when PYK2 knockout samples were examined. Wild-type and PYK2 knockout samples were subjected to SDS-PAGE. ab32571 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PYK2 antibody [YE353] (ab32571)

Predicted band size: 116 kDa

false

• WB

Lab

Western blot - Anti-PYK2 antibody [YE353] (AB32571)

Western blot : Anti-PTK2B antibody [YE353] (ab32571) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab32571 was shown to bind specifically to PTK2B. A band was observed at 130 kDa in wild-type A549 cell lysates with no signal observed at this size in PTK2B knockout cell line. To generate this image, wild-type and PTK2B 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-PYK2 antibody [YE353] (ab32571) at 1/2000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

PTK2B knockout A549 cell lysate at 20 µg

Lane 3:

Wild-type HAP1 cell lysate at 20 µg

Lane 4:

PTK2B knockout HAP1 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: 130 kDa

false

• WB

Lab

Western blot - Anti-PYK2 antibody [YE353] (AB32571)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-PYK2 antibody [YE353] (ab32571) at 1/10000 dilution

Lane 1:

Ramos cell lysate at 20 µg

Lane 2:

Jurkat cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 116 kDa

Observed band size: 116 kDa

false

• WB

Lab

Western blot - Anti-PYK2 antibody [YE353] (AB32571)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-PYK2 antibody [YE353] (ab32571) at 1/2000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Rat brain tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 116 kDa

Observed band size: 116 kDa

false

• WB

Lab

Western blot - Anti-PYK2 antibody [YE353] (AB32571)

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Western blot - Anti-PYK2 antibody [YE353] (ab32571) at 10000 Cells

All lanes:

RAW264.7 cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 116 kDa

Observed band size: 116 kDa

false

• WB

Unknown

Western blot - Anti-PYK2 antibody [YE353] (AB32571)

All lanes:

Western blot - Anti-PYK2 antibody [YE353] (ab32571) at 1/5000 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 116 kDa

Observed band size: 116 kDa

false