Human PODXL knockout HCT116 cell lysate (ab257211)
Overview
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Product name
Human PODXL knockout HCT116 cell lysate
See all PODXL kits -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HCT116 -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion in exon2. -
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 ab263498 - Human PODXL knockout HCT116 cell lysate 1 x 100µg ab255555 - Human wild-type HCT116 cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
Disease
Carcinoma -
STR Analysis
Amelogenin X D5S818: 10, 11 D13S317: 10, 12 D7S820: 11, 12 D16S539: 11, 13 vWA: 17, 22 TH01: 8,9 TPOX: 8, 9 CSF1PO: 7, 10
Target
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Function
Involved in the regulation of both adhesion and cell morphology and cancer progression. Function as an anti-adhesive molecule that maintains an open filtration pathway between neighboring foot processes in the podocyte by charge repulsion. Acts as a pro-adhesive molecule, enhancing the adherence of cells to immobilized ligands, increasing the rate of migration and cell-cell contacts in an integrin-dependent manner. Induces the formation of apical actin-dependent microvilli. Involved in the formation of a preapical plasma membrane subdomain to set up inital epithelial polarization and the apical lumen formation during renal tubulogenesis. Plays a role in cancer development and aggressiveness by inducing cell migration and invasion through its interaction with the actin-binding protein EZR. Affects EZR-dependent signaling events, leading to increased activities of the MAPK and PI3K pathways in cancer cells. -
Tissue specificity
Glomerular epithelium cell (podocyte). -
Sequence similarities
Belongs to the podocalyxin family. -
Domain
Both the O-glycan-rich domain of the extracellular domain and the C-terminus PDZ-binding motif (DTHL) in the cytoplasmic tail harbor an apical sorting signal. The cytoplasmic domain is necessary for the apical membrane targeting and renal tubulogenesis. The cytoplasmic C-terminus PDZ-binding motif (DTHL) is essential for interaction with SLC9A3R1 and for targeting SLC9A3R1 to the apical cell membrane. The extracellular domain is necessary for microvillus formation (By similarity). The large highly anionic extracellular domain allows to maintain open filtration pathways between neighboring podocyte foot processes. -
Post-translational
modificationsN- and O-linked glycosylated. Sialoglycoprotein. -
Cellular localization
Apical cell membrane. Cell projection, lamellipodium. Cell projection, filopodium. Cell projection, ruffle. Cell projection, microvillus. Membrane raft. Membrane. In single attached epithelial cells is restricted to a preapical pole on the free plasma membrane whereas other apical and basolateral proteins are not yet polarized. Colocalizes with SLC9A3R2 at the apical plasma membrane during epithelial polarization. Colocalizes with SLC9A3R1 at the trans-Golgi network (transiently) and at the apical plasma membrane. Its association with the membrane raft is transient. Colocalizes with actin filaments, EZR and SLC9A3R1 in a punctate pattern at the apical cell surface where microvilli form. Colocalizes with EZR and SLC9A3R2 at the apical cell membrane of glomerular epithelium cells (By similarity). Forms granular, punctuated pattern, forming patches, preferentially adopting a polar distribution, located on the migrating poles of the cell or forming clusters along the terminal ends of filipodia establishing contact with the endothelial cells. Colocalizes with the submembrane actin of lamellipodia, particularly associated with ruffles. Colocalizes with vinculin at protrusions of cells. Colocalizes with ITGB1. Colocalizes with PARD3, PRKCI, EXOC5, OCLN, RAB11A and RAB8A in apical membrane initiation sites (AMIS) during the generation of apical surface and luminogenesis (By similarity). - Information by UniProt
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Form
There are 2 isoforms produced by alternative splicing. -
Alternative names
- GCTM-2 antigen
- Gp2
- Gp200
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 ab257211 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 |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 58 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 58 kDa. |
Images
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Lane 1: Wild-type HCT116 cell lysate (20µg)
Lane 2: PODXL knockout HCT116 cell lysate (20µg)
Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 200 kDa. Red - loading control ab7291 observed at 50 kDa.
ab150358 Anti-PODXL antibody [EPR9518] was shown to specifically react with PODXL in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266887 (knockout cell lysate ab257211) was used. Wild-type and PODXL 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. ab150358 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution 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. -
Homozygous: 1 bp insertion in exon2
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)
ab257211 has not yet been referenced specifically in any publications.