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AB266504

Human ZYX (Zyxin) knockout HEK-293T cell line

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ZYX KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 2.

View Alternative Names

ESP 2, HED 2, ZYX protein, ZYX_HUMAN, Zyxin, Zyxin-2

4 Images
Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)
  • WB

Lab

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)

Lanes 1 - 4 : Merged signal (red and green). Green - ab50391 observed at 75 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37 kDa.

ab50391 was shown to react with ZYX in wild-type HEK-293T cells in Western blot with loss of signal observed in ZYX knockout cell line ab266504 (ZYX knockout cell lysate ab257810). Wild-type HEK-293T and ZYX knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab50391 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Zyxin antibody [ZOL301] (<a href='/en-us/products/primary-antibodies/zyxin-antibody-zol301-ab50391'>ab50391</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

ZYX knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (ab266504)

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

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Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)
  • WB

Lab

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)

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] (<a href='/en-us/products/primary-antibodies/zyxin-antibody-epr4302-ab109316'>ab109316</a>) 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 (ab266504)

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

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Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)
  • WB

Lab

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)

Lanes 1 - 4 : Merged signal (red and green). Green - ab229757 observed at 75 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab229757 was shown to react with ZYX in wild-type HEK-293T cells in Western blot with loss of signal observed in ZYX knockout cell line ab266504 (ZYX knockout cell lysate ab257810). Wild-type HEK-293T and ZYX knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab229757 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-Zyxin antibody - N-terminal (<a href='/en-us/products/primary-antibodies/zyxin-antibody-n-terminal-ab229757'>ab229757</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

ZYX knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human ZYX (Zyxin) knockout HEK-293T cell line (ab266504)

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

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Sanger Sequencing - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)
  • Sanger seq

Unknown

Sanger Sequencing - Human ZYX (Zyxin) knockout HEK-293T cell line (AB266504)

Homozygous : Insertion of the selection cassette in exon 2

Key facts

Cell type

HEK-293T

Species or organism

Human

Tissue

Kidney

Form

Liquid

form

Knockout validation

Sanger Sequencing,Western blot

Mutation description

Knockout achieved by using CRISPR/Cas9, Homozygous: Insertion of the selection cassette in exon 2

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Reactivity data

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Product details

Recommended control: Human wild-type HEK293T cell line (ab255449). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.

We will provide viable cells that proliferate on revival.

Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.

This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

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What's included?

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Properties and storage information

Gene name
ZYX
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Sanger Sequencing, Western blot
Zygosity
Homozygous
Shipped at conditions
Dry Ice
Appropriate short-term storage conditions
-196°C
Appropriate long-term storage conditions
-196°C
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Handling procedures

Initial handling guidelines

Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.

1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.

Subculture guidelines
  • All seeding densities should be based on cell counts gained by established methods.
  • A guide seeding density of 2x104 cells/cm2 is recommended.
  • Cells should be passaged when they have achieved 80-90% confluence.
Culture medium

DMEM (High Glucose) + 10% FBS

Cryopreservation medium

Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

Zyxin also known as ZYX is a mechanical regulator associated with cell adhesion and signaling. This protein has a molecular mass of approximately 61 kDa and shows expression in various cell types including those in the heart liver and lungs. As a component of focal adhesions zyxin plays a role in cytoskeletal dynamics by interacting with actin filaments and facilitating the formation of stress fibers.
Biological function summary

The protein has important roles in cellular processes such as migration and morphology. Zyxin often forms part of a larger protein complex that includes VASP and α-actinin which contribute to cell scaffolding and structural integrity. It serves as an organizer for cytoskeletal proteins ensuring proper cell movement and adaptability to cellular stress. Its actin-binding capacity enables it to regulate structural changes necessary for cellular adaptation to environmental stimuli.

Pathways

This protein is essential in the regulation of actin cytoskeleton associated pathways and adherens junction signaling. Zyxin interacts with Ena/VASP proteins which are significant in actin filament elongation helping regulate dynamic changes in cell shape and motility. Its relationship with LIM domain-containing proteins positions zyxin within pathways related to cell proliferation and signal transduction.

Zyxin has been implicated in cancer progression especially in metastasis where altered cell adhesion and movement play a role. It also relates to cardiovascular diseases due to its involvement in mechanotransduction pathways critical to heart muscle function. In cancer zyxin influences the activity of YAP/TAZ transcription regulators affecting tumor cell behavior. In cardiovascular scenarios the protein interacts with paxillin to mediate responses to mechanical load changes impacting heart disease progression.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 2 US: 2

Adherent/suspension

Adherent

Gender

Female

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Product protocols

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Target data

See full target information ZYX
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Primary Antibodies

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