Anti-Zyxin antibody [EPR4302]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Zyxin antibody [EPR4302] (AB109316)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Zyxin with Purified ab109316 at 1/100 dilution (10 μg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Zyxin antibody [EPR4302] (AB109316)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Zyxin with Purified ab109316 at 1/500 dilution (1.87 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. 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. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Zyxin antibody [EPR4302] (AB109316)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human gastric carcinoma tissue sections labeling Zyxin with purified ab109316 at 1/1000 dilution (0.94 μg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Zyxin antibody [EPR4302] (AB109316)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling Zyxin with purified ab109316 at 1/1000 dilution (0.94 μg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Zyxin antibody [EPR4302] (AB109316)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat kidney tissue sections labeling Zyxin with purified ab109316 at 1/1000 dilution (0.94 μg/mL). Heat mediated antigen retrieval using Bond™™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Unknown

Immunoprecipitation - Anti-Zyxin antibody [EPR4302] (AB109316)

Purified ab109316 at 1/50 dilution (2ug) immunoprecipitating Zyxin in Mouse testis lysate.
Lane 1 (input) : Mouse testis lysate (10μg)
Lane 2 (+) : ab109316 + Mouse testis lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109316 in mouse testis lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 82 kDa
We are unsure about the extra bands. They might be the cleavage fragments as what are described in PMID : 17572661

All lanes:

Immunoprecipitation - Anti-Zyxin antibody [EPR4302] (ab109316)

Predicted band size: 61 kDa

false

• WB

Unknown

Western blot - Anti-Zyxin antibody [EPR4302] (AB109316)

Blocking/Diluting buffer : 5% NFDM/TBST

We are unsure about the extra bands. They might be the cleavage fragments as what are described in PMID : 17572661

All lanes:

Western blot - Anti-Zyxin antibody [EPR4302] (ab109316) at 1/20000 dilution

All lanes:

HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 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

Predicted band size: 61 kDa

Observed band size: 82 kDa

false

• WB

Lab

Western blot - Anti-Zyxin antibody [EPR4302] (AB109316)

False colour image of Western blot : Anti-Zyxin antibody [EPR4302] staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109316 was shown to bind specifically to Zyxin. A band was observed at 75 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in ZYX knockout cell line ab266503 (knockout cell lysate ab257809). The band observed in the knockout lysate lane below 75 kDa is likely to represent a truncated form of Zyxin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZYX knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Zyxin antibody [EPR4302] (ab109316) at 1/20000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

ZYX knockout HEK-293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 61 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-Zyxin antibody [EPR4302] (AB109316)

False colour image of Western blot : Anti-Zyxin antibody [EPR4302] staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109316 was shown to bind specifically to Zyxin. A band was observed at 75 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in ZYX knockout cell line ab266504 (knockout cell lysate ab257810). The band observed in the knockout lysate lane below 75 kDa is likely to represent a truncated form of Zyxin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZYX knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Zyxin antibody [EPR4302] (ab109316) at 1/20000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

ZYX knockout HEK-293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 61 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-Zyxin antibody [EPR4302] (AB109316)

False colour image of Western blot : Anti-Zyxin antibody [EPR4302] staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109316 was shown to bind specifically to Zyxin. A band was observed at 75 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in ZYX CRISPR-Cas9 edited cell line ab266504 (CRISPR-Cas9 edited cell lysate ab257810). The band observed in the CRISPR-Cas9 edited lysate lane below 75 kDa is likely to represent a truncated form of Zyxin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZYX CRISPR-Cas9 edited HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Zyxin antibody [EPR4302] (ab109316) at 1/20000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

ZYX CRISPR-Cas9 edited HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-zyx-zyxin-knockout-hek-293t-cell-line-ab266504'>ab266504</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 61 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-Zyxin antibody [EPR4302] (AB109316)

False colour image of Western blot : Anti-Zyxin antibody [EPR4302] staining at 1/20000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109316 was shown to bind specifically to Zyxin. A band was observed at 75 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in ZYX CRISPR-Cas9 edited cell line ab266503 (CRISPR-Cas9 edited cell lysate ab257809). The band observed in the CRISPR-Cas9 edited lysate lane below 75 kDa is likely to represent a truncated form of Zyxin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZYX CRISPR-Cas9 edited HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Zyxin antibody [EPR4302] (ab109316) at 1/20000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

ZYX CRISPR-Cas9 edited HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-zyx-zyxin-knockout-hek-293t-cell-line-ab266503'>ab266503</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

Daudi cell lysate at 20 µg

Predicted band size: 61 kDa

Observed band size: 75 kDa

false

• WB

Unknown

Western blot - Anti-Zyxin antibody [EPR4302] (AB109316)

Blocking/Diluting buffer : 5% NFDM/TBST

We are unsure about the extra bands. They might be the cleavage fragments as what are described in PMID : 17572661

All lanes:

Western blot - Anti-Zyxin antibody [EPR4302] (ab109316) at 1/20000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

C2C12 (Mouse myoblasts myoblast) whole cell lysates at 20 µg

Lane 3:

Mouse lung lysates at 20 µg

Lane 4:

Mouse testis lysates at 20 µg

Lane 5:

Rat lung lysates at 20 µg

Lane 6:

Rat testis lysates at 20 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

false