Human RHOA knockout HEK-293T cell lysate (ab257637)
Overview
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Product name
Human RHOA knockout HEK-293T cell lysate
See all RhoA kits -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 4 bp insertion in exon 2 and Insertion of the selection cassette in exon 2. -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.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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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab260309 - Human RHOA knockout HEK293T cell lysate 1 x 100µg ab255553 - Human wild-type HEK293T cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
STR Analysis
Amelogenin X D5S818: 8, 9 D13S317: 12, 14 D7S820: 11 D16S539: 9, 13 vWA: 16, 19 TH01: 7, 9.3 TPOX: 11 CSF1PO: 11, 12
Target
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Function
Regulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Serves as a target for the yopT cysteine peptidase from Yersinia pestis, vector of the plague, and Yersinia pseudotuberculosis, which causes gastrointestinal disorders. May be an activator of PLCE1. Activated by ARHGEF2, which promotes the exchange of GDP for GTP. -
Sequence similarities
Belongs to the small GTPase superfamily. Rho family. -
Domain
The basic-rich region is essential for yopT recognition and cleavage. -
Post-translational
modificationsSubstrate for botulinum ADP-ribosyltransferase.
Cleaved by yopT protease when the cell is infected by some Yersinia pathogens. This removes the lipid attachment, and leads to its displacement from plasma membrane and to subsequent cytoskeleton cleavage.
AMPylation at Tyr-34 and Thr-37 are mediated by bacterial enzymes in case of infection by H.somnus and V.parahaemolyticus, respectively. AMPylation occurs in the effector region and leads to inactivation of the GTPase activity by preventing the interaction with downstream effectors, thereby inhibiting actin assembly in infected cells. It is unclear whether some human enzyme mediates AMPylation; FICD has such ability in vitro but additional experiments remain to be done to confirm results in vivo.
Ubiquitinated by the BCR(BACURD1) and BCR(BACURD2) E3 ubiquitin ligase complexes, leading to its degradation by the proteasome, thereby regulating the actin cytoskeleton and cell migration. -
Cellular localization
Cell membrane. Cytoplasm > cytoskeleton. - Information by UniProt
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Alternative names
- Aplysia ras related homolog 12
- ARH12
- ARHA
see all
Associated products
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KO cell lines
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab257637 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 22 kDa.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 22 kDa. |
Images
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Western blot - Human RHOA knockout HEK293T cell lysate (ab257637)Lane 1: Wild-type HEK-293T cell lysate (20µg)
Lane 2: RHOA knockout HEK-293T cell lysate (20µg)
Lane 3: Wild-type HEK-293T cell lysate (20µg)
Lane 4: RHOA knockout HEK-293T cell lysate (20µg)
Lanes 1- 4: Merged signal (red and green). Green - ab187027 observed at 21 kDa. Red - loading control ab7291 observed at 50 kDa.
ab187027 Anti-RhoA antibody [EPR18134] was shown to specifically react with RhoA in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266592 (knockout cell lysate ab257637) was used. Wild-type and RhoA knockout samples were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab187027 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 5000 and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging. -
Sanger Sequencing - Human RHOA knockout HEK293T cell lysate (ab257637)Allele-1: 4 bp insertion in exon 2
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Sanger Sequencing - Human RHOA knockout HEK293T cell lysate (ab257637)Allele-2: Insertion of the selection cassette in exon 2
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab257637 has not yet been referenced specifically in any publications.