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  1. Link

    human-ikbkg-ikk-gammanemo-knockout-hek-293t-cell-lysate-ab257153.pdf

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Cell Biology Apoptosis Intracellular NFkB IKK
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Human IKBKG (IKK gamma/NEMO) knockout HEK-293T cell lysate (ab257153)

  • Datasheet
  • SDS
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Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK293T cell lysate (ab257153)
  • Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK293T cell lysate (ab257153)
  • Sanger Sequencing - Human IKBKG knockout HEK293T cell lysate (ab257153)
  • Sanger Sequencing - Human IKBKG knockout HEK293T cell lysate (ab257153)

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Primary
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Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872)
Primary
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Anti-IKK gamma/NEMO antibody [EPR14660] (ab188569)
Knockout
Product image
Human ARHGDIA (RhoGDI) knockout HEK-293T cell lysate (ab257356)

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Overview

  • Product name

    Human IKBKG (IKK gamma/NEMO) knockout HEK-293T cell lysate
    See all IKK gamma/NEMO kits
  • Product overview

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

  • Parental Cell Line

    HEK293T
  • Organism

    Human
  • Mutation description

    Knockout achieved by using CRISPR/Cas9, 13 bp deletion in exon 2 and 1 bp insertion in exon 2.
  • Passage number

    <20
  • Knockout validation

    Sanger Sequencing
  • 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.

  • 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.

  • Tested applications

    Suitable for: WBmore details

Properties

  • Storage instructions

    Store at -80°C. Please refer to protocols.
  • Components 1 kit
    ab260190 - Human IKBKG knockout HEK293T cell lysate 1 x 100µg
    ab255553 - Human wild-type HEK293T cell lysate 1 x 100µg
  • Research areas

    • Cell Biology
    • Apoptosis
    • Intracellular
    • NFkB
    • IKK
    • Signal Transduction
    • Protein Phosphorylation
    • Ser / Thr Kinases
    • Other Kinases
    • Signal Transduction
    • Signaling Pathway
    • Nuclear Signaling
    • NFkB Pathway
    • Epigenetics and Nuclear Signaling
    • Nuclear Signaling Pathways
    • NFkB pathway
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Inflammatory mediators
    • Immunology
    • Innate Immunity
    • TLR Signaling
    • Cancer
    • Cell Death
    • Apoptosis
    • Apoptosis Markers
    • NFkB
    • IKK
  • 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

  • Function

    Regulatory subunit of the IKK core complex which phosphorylates inhibitors of NF-kappa-B thus leading to the dissociation of the inhibitor/NF-kappa-B complex and ultimately the degradation of the inhibitor. Also considered to be a mediator for TAX activation of NF-kappa-B. Could be implicated in NF-kappa-B-mediated protection from cytokine toxicity (By similarity). Essential for viral activation of IRF3.
  • Tissue specificity

    Heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
  • Involvement in disease

    Defects in IKBKG are the cause of ectodermal dysplasia anhidrotic with immunodeficiency X-linked (EDAID) [MIM:300291]; also known as hypohidrotic ectodermal dysplasia with immunodeficiency (HED-ID). Is a form of ectoderma dysplasia, a heterogeneous group of disorders due to abnormal development of two or more ectodermal structures. Characterized by absence of sweat glands, sparse scalp hair, rare conical teeth and immunological abnormalities resulting in severe infectious diseases.
    Defects in IKBKG are the cause of ectodermal dysplasia anhidrotic with immunodeficiency-osteopetrosis-lymphedema (OLEDAID) [MIM:300301].
    Defects in IKBKG are a cause of immunodeficiency NEMO-related without anhidrotic ectodermal dysplasia (NEMOID) [MIM:300584]; also called immunodeficiency without anhidrotic ectodermal dysplasia, isolated immunodeficiency or pure immunodeficiency. Patients manifest immunodeficiency not associated with other abnormalities, and resulting in increased infection susceptibility. Patients suffer from multiple episodes of infectious diseases.
    Defects in IKBKG are the cause of susceptibility to X-linked familial atypical micobacteriosis type 1 (AMCBX1) [MIM:300636]; also known as X-linked disseminated atypical mycobacterial infection type 1 or X-linked susceptibility to mycobacterial disease type 1. AMCBX1 is the X-linked recessive form of mendelian susceptibility to mycobacterial disease (MSMD). MSMD is a congenital syndrome resulting in predisposition to clinical disease caused by weakly virulent mycobacterial species, such as bacillus Calmette-Guerin vaccines and non-tuberculous, environmental mycobacteria. Patients are also susceptible to the more virulent species Mycobacterium tuberculosis.
    Defects in IKBKG are the cause of recurrent isolated invasive pneumococcal disease type 2 (IPD2) [MIM:300640]. Recurrent invasive pneumococcal disease (IPD) is defined as two episodes of IPD occurring at least 1 month apart, whether caused by the same or different serotypes or strains. Recurrent IPD occurs in at least 2% of patients in most series, making IPD the most important known risk factor for subsequent IPD.
    Defects in IKBKG are the cause of incontinentia pigmenti (IP) [MIM:308300]; formerly designed familial incontinentia pigmenti type II (IP2). IP is a genodermatosis usually prenatally lethal in males. In affected females, it causes abnormalities of the skin, hair, eyes, nails, teeth, skeleton, heart, and central nervous system. The prominent skin signs occur in four classic cutaneous stages: perinatal inflammatory vesicles, verrucous patches, a distinctive pattern of hyperpigmentation and dermal scarring.
  • Sequence similarities

    Contains 1 C2HC-type zinc finger.
  • Domain

    The leucine-zipper domain and the C2HC-type zinc-finger are essential for polyubiquitin binding and for the activation of IRF3.
  • Post-translational
    modifications

    Phosphorylation at Ser-68 attenuates aminoterminal homodimerization.
    Polyubiquitinated on Lys-285 through 'Lys-63'; the ubiquitination is mediated by NOD2 and RIPK2 and probably plays a role in signaling by facilitating interactions with ubiquitin domain-containing proteins and activates the NF-kappa-B pathway. Polyubiquitinated on Lys-399 through 'Lys-63'; the ubiquitination is mediated by BCL10, MALT1 and TRAF6 and probably plays a role in signaling by facilitating interactions with ubiquitin domain-containing proteins and activates the NF-kappa-B pathway. Monoubiquitinated on Lys-277 and Lys-309; promotes nuclear export. Linear polyubiquitinated on Lys-285; the head-to-tail polyubiquitination is mediated by the LUBAC complex. Linear polyubiquitinated on Lys-309; the head-to-tail polyubiquitination is mediated by the LUBAC complex.
    Sumoylated on Lys-277 and Lys-309 by SUMO1; the modification results in phosphorylation of Ser-85 by ATM leading to a replacement of the sumoylation by mono-ubiquitination on these residues.
  • Cellular localization

    Cytoplasm. Nucleus. Sumoylated NEMO accumulates in the nucleus in response to genotoxic stress.
  • Target information above from: UniProt accession Q9Y6K9 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names

    • IkB kinase associated protein 1
    • IkB kinase subunit gamma
    • Inhibitor of nuclear factor kappa B kinase subunit gamma
    • AMCBX1
    • FIP 3
    • FIP-3
    • FIP3
    • Fip3p
    • I kappa B kinase gamma
    • I-kappa-B kinase subunit gamma
    • IkB kinase gamma subunit
    • IkB kinase subunit gamma
    • IkB kinase-associated protein 1
    • Ikbkg
    • IKK gamma
    • IKK-gamma
    • IKKAP1
    • IKKG
    • IMD33
    • Incontinentia pigmenti
    • Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase gamma
    • Inhibitor of kappa light polypeptide gene enhancer in B cells, kinase of, gamma
    • Inhibitor of nuclear factor kappa-B kinase subunit gamma
    • IP
    • IP1
    • IP2
    • IPD2
    • NEMO
    • NEMO_HUMAN
    • NF kappa B essential modifier
    • NF kappa B essential modulator
    • NF-kappa-B essential modifier
    • NF-kappa-B essential modulator
    • ZC2HC9
    see all

Associated products

  • KO cell lines

    • Human IKBKG (IKK gamma/NEMO) knockout HEK-293T cell line (ab266674)
  • Related Products

    • Anti-IKK gamma/NEMO antibody [EPR16629] (ab178872)
    • Anti-IKK gamma/NEMO antibody [EPR14660] (ab188569)
    • Anti-IKK gamma/NEMO antibody [EPR16629] - BSA and Azide free (ab230832)
    • Anti-IKK gamma/NEMO antibody [EPR14660] - BSA and Azide free (ab250964)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab257153 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: 48 kDa.

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

Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 48 kDa.

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

Images

  • Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK293T cell lysate (ab257153)
    Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK293T cell lysate (ab257153)
    Lane 1: Wild-type HEK-293T cell lysate (20µg)

    Lane 2: IKBKG knockout HEK-293T cell lysate (20µg)

    Lanes 1- 2: Merged signal (red and green). Green - ab188569 observed at 48 kDa. Red - loading control, ab8245 observed at 37 kDa.

    ab188569 Anti-IKK gamma/NEMO antibody [EPR14660] was shown to specifically react with IKK gamma/NEMO in wild-type HEK-293T cells in western blot. The band observed in the knockout cell line ab266674 (knockout cell lysate ab257153) lane below 48kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and IKK gamma/NEMO 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. ab188569 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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.
  • Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK293T cell lysate (ab257153)
    Western blot - Human IKBKG (IKK gamma/NEMO) knockout HEK293T cell lysate (ab257153)
    Lane 1: Wild-type HEK-293T cell lysate (20µg)

    Lane 2: IKBKG knockout HEK-293T cell lysate (20µg)

    Lanes 1- 2: Merged signal (red and green). Green - ab178872 observed at 48 kDa. Red - loading control, ab8245 observed at 37 kDa.

    ab178872 Anti-IKK gamma/NEMO antibody [EPR16629] was shown to specifically react with IKK gamma/NEMO in wild-type HEK-293T cells in western blot. The band observed in the knockout cell line ab266674 (knockout cell lysate ab257153) lane below 48kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and IKK gamma/NEMO 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. ab178872 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 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 IKBKG knockout HEK293T cell lysate (ab257153)
    Sanger Sequencing - Human IKBKG knockout HEK293T cell lysate (ab257153)

    Allele-1: 13 bp deletion in exon 2

     

  • Sanger Sequencing - Human IKBKG knockout HEK293T cell lysate (ab257153)
    Sanger Sequencing - Human IKBKG knockout HEK293T cell lysate (ab257153)

    Allele-2: 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.

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (0)

Publishing research using ab257153? Please let us know so that we can cite the reference in this datasheet.

ab257153 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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Abreviews
Abreviews
abreview image
NEMO/IKKy has a tricky band pattern on blots, at least with the abcam ab178872 primary. When I first probed for NEMO with ab178872 (before I received the lysates), I got a lot of bands that were ambiguous: which one is NEMO? Then I ordered the sample KO lysate and ran it in parallel with my own samples. the NEMO band was obvious, allowing me to verify an siNEMO I want to use.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

Ms. Olivia Harding

Verified customer

Submitted May 07 2021

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