Human DAG1 knockout HEK-293T cell lysate (ab257192)
Overview
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Product name
Human DAG1 knockout HEK-293T cell lysate -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HEK293T -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp insertion 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 ab260173 - Human DAG1 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
The dystroglycan complex is involved in a number of processes including laminin and basement membrane assembly, sarcolemmal stability, cell survival, peripheral nerve myelination, nodal structure, cell migration, and epithelial polarization.
Alpha-dystroglycan is an extracellular peripheral glycoprotein that acts as a receptor for both extracellular matrix proteins containing laminin-G domains, and for certain adenoviruses. Receptor for laminin-2 (LAMA2) and agrin in peripheral nerve Schwann cells. Also acts as a receptor for M.leprae in peripheral nerve Schwann cells but only in the presence of the G-domain of LAMA2, and for lymphocytic choriomeningitis virus, Old World Lassa fever virus, and clade C New World arenaviruses.
Beta-dystroglycan is a transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton. Acts as a cell adhesion receptor in both muscle and non-muscle tissues. Receptor for both DMD and UTRN and, through these interactions, scaffolds axin to the cytoskeleton. Also functions in cell adhesion-mediated signaling and implicated in cell polarity. -
Tissue specificity
Expressed in a variety of fetal and adult tissues. In epidermal tissue, located to the basement membrane. Also expressed in keratinocytes and fibroblasts. -
Involvement in disease
Defects in DAG1 are the cause of muscular dystrophy-dystroglycanopathy limb-girdle type C7 (MDDGC7) [MIM:613818]. An autosomal recessive muscular dystrophy showing onset in early childhood, and associated with mental retardation without structural brain anomalies. Note=MDDGC7 is caused by DAG1 mutations that interfere with normal post-translational processing, resulting in defective DAG1 glycosylation and impaired interactions with extracellular-matrix components. Other muscular dystrophy-dystroglycanopathies are caused by defects in enzymes involved in protein O-glycosylation. -
Sequence similarities
Contains 1 peptidase S72 domain. -
Post-translational
modificationsO- and N-glycosylated. Alpha-dystroglycan is heavily O-glycosylated comprising of up to two thirds of its mass and the carbohydrate composition differs depending on tissue type. Mucin-type O-glycosylation is important for ligand binding activity. O-mannosylation of alpha-DAG1 is found in high abundance in both brain and muscle where the most abundant glycan is Sia-alpha-2-3-Gal-beta-1-4-Glc-NAc-beta-1-2-Man. In muscle, glycosylation on Thr-379 by a phosphorylated O-mannosyl glycan with the structure 2-(N-acetylamido)-2-deoxygalactosyl-beta-1,3-2-(N-acetylamido)-2-deoxyglucosyl-beta-1,4-6-phosphomannose is mediated by like-acetylglucosaminyltransferase (LARGE) protein and is required for laminin binding. O-mannosylation is also required for binding lymphocytic choriomeningitis virus, Old World Lassa fever virus, and clade C New World arenaviruses. The O-glycosyl hexose on Thr-367, Thr-369, Thr-372, Thr-381 and Thr-388 is probably mannose. O-glycosylated in the N-terminal region with a core 1 or possibly core 8 glycan. The beta subunit is N-glycosylated.
Autolytic cleavage produces the alpha and beta subunits. In cutaneous cells, as well as in certain pathological conditions, shedding of beta-dystroglcan can occur releasing a peptide of about 30 kDa.
SRC-mediated phosphorylation of the PPXY motif of the beta subunit recruits SH2 domain-containing proteins, but inhibits binding to WWW domain-containing proteins, DMD and UTRN. This phosphorylation also inhibits nuclear entry. -
Cellular localization
Secreted > extracellular space and Cell membrane. Cytoplasm > cytoskeleton. Nucleus > nucleoplasm. The monomeric form translocates to the nucleus via the action of importins and depends on RAN. Nuclear transport is inhibited by Tyr-892 phosphorylation. In skeletal muscle, this phosphorylated form locates to a vesicular internal membrane compartment. In peripheral nerves, localizes to the Schwann cell membrane. Colocalizes with ERM proteins in Schwann-cell microvilli. - Information by UniProt
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Alternative names
- 156DAG
- A3a
- Agrin receptor
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 ab257192 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: 97 kDa.
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Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 97 kDa. |
Images
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Lane 1: Wild-type HEK293T cell lysate (20 µg)
Lane 2: DAG1 knockout HEK293T cell lysate (20 µg)
Lane 3: Mouse heart tissue lysate (20 µg)
Lanes 1-3: Merged signal (red and green). Green - ab62373 observed at 43 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab62373 Anti-beta Dystroglycan antibody [EP2200Y] was shown to specifically react with beta Dystroglycan in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266263 (knockout cell lysate ab257192) was used. Wild-type and beta Dystroglycan knockout samples were subjected to SDS-PAGE. ab62373 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 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 10000 dilution for 1 hour at room temperature before imaging.
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Lane 1: Wild-type HEK293T cell lysate (20 µg)
Lane 2: DAG1 knockout HEK293T cell lysate (20 µg)
Lane 3: Human skeletal muscle tissue lysate (20 µg)
Lanes 1-3: Merged signal (red and green). Green - ab43125 observed at 43 kDa. Red - loading control, ab8245 observed at 37 kDa.
ab43125 Anti-beta Dystroglycan antibody was shown to specifically react with beta Dystroglycan in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266263 (knockout cell lysate ab257192) was used. Wild-type and beta Dystroglycan knockout samples were subjected to SDS-PAGE. ab43125 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 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 10000 dilution for 1 hour at room temperature before imaging.
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Homozygous: 1 bp insertion 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)
ab257192 has not yet been referenced specifically in any publications.