Validated using a knockout cell line
Recombinant
RabMAb

Recombinant Anti-GCLM antibody [EPR6667] (ab126704)

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Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR6667] to GCLM
  • Suitable for: WB, IP, IHC-P, Flow Cyt
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Overview

  • Product name

    Anti-GCLM antibody [EPR6667]
    See all GCLM primary antibodies

  • Description

    Rabbit monoclonal [EPR6667] to GCLM

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IP, IHC-P, Flow Cytmore details
    Unsuitable for: ICC

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide within Human GCLM aa 50-150. The exact sequence is proprietary.

  • Positive control

    • HeLa, A673, PC12, A431, and K562 cell lysates; Human breast carcinoma tissue.

  • General notes

     

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Applications

Our Abpromise guarantee covers the use of ab126704 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 1/1000 - 1/10000. Predicted molecular weight: 31 kDa.
IP 1/10 - 1/100.
IHC-P 1/50 - 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt 1/10 - 1/100.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
  • Application notes
    Is unsuitable for ICC.

    • Target

    • Tissue specificity

      In all tissues examined. Highest levels in skeletal muscle.

    • Pathway

      Sulfur metabolism; glutathione biosynthesis; glutathione from L-cysteine and L-glutamate: step 1/2.

    • Sequence similarities

      Belongs to the aldo/keto reductase family. Glutamate--cysteine ligase light chain subfamily.
    • Information by UniProt

    • Database links

      see all

    • Alternative names

      • Gamma ECS regulatory subunit antibody
      • Gamma-ECS regulatory subunit antibody
      • Gamma-glutamylcysteine synthetase regulatory subunit antibody
      • GCLM antibody
      • GCS light chain antibody
      • GLCLR antibody
      • Glutamate cysteine ligase regulatory subunit antibody
      • Glutamate--cysteine ligase modifier subunit antibody
      • Glutamate--cysteine ligase regulatory subunit antibody
      • GSC light chain antibody
      • GSH0_HUMAN antibody
      see all

    Images

    • Western blot - Anti-GCLM antibody [EPR6667] (ab126704)
      Western blot - Anti-GCLM antibody [EPR6667] (ab126704)
      All lanes : Anti-GCLM antibody [EPR6667] (ab126704) at 1 µg/ml

      Lane 1 : Wild-type HAP1 whole cell lysate
      Lane 2 : GCLM knockout HAP1 whole cell lysate
      Lane 3 : HeLa whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Predicted band size: 31 kDa



      Lanes 1 - 3: Merged signal (red and green). Green - ab126704 observed at 31 kDa. Red - loading control, ab130007, observed at 130 kDa.

      ab126704 was shown to recognize GCLM in wild-type HAP1 cells as signal was lost at the expected MW in GCLM knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GCLM knockout samples were subjected to SDS-PAGE. Ab126704 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed 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 1/20000 dilution for 1 hour at room temperature before imaging.

    • Flow Cytometry - Anti-GCLM antibody [EPR6667] (ab126704)
      Flow Cytometry - Anti-GCLM antibody [EPR6667] (ab126704)
      Overlay histogram showing HeLa cells stained with ab126704 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab126704, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCLM antibody [EPR6667] (ab126704)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GCLM antibody [EPR6667] (ab126704)

      ab126704, at 1/50 dilution, staining GCLM in Paraffin-embedded Human breast carcinoma tissue by Immunohistochemistry.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Immunoprecipitation - Anti-GCLM antibody [EPR6667] (ab126704)
      Immunoprecipitation - Anti-GCLM antibody [EPR6667] (ab126704)

      Purified ab126704 at 1/20 dilution (0.6µg) immunoprecipitating GCLM in HeLa whole cell lysate.
      Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
      Lane 2 (+): ab126704 + HeLa whole cell lysate.
      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab126704 in HeLa whole cell lysate.
      VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/5000 dilution) was used for Western blotting.
      Blocking Buffer and concentration: 5% NFDM/TBST.
      Diluting buffer and concentration: 5% NFDM/TBST.
      Observed band size: 31 kDa

    • Western blot - Anti-GCLM antibody [EPR6667] (ab126704)
      Western blot - Anti-GCLM antibody [EPR6667] (ab126704)
      All lanes : Anti-GCLM antibody [EPR6667] (ab126704) at 1/1000 dilution

      Lane 1 : HeLa cell lysate
      Lane 2 : A673 cell lysate
      Lane 3 : PC12 cell lysate
      Lane 4 : A431 cell lysate
      Lane 5 : K562 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat anti-Rabbit HRP at 1/2000 dilution

      Predicted band size: 31 kDa

    References

    Publishing research using ab126704? Please let us know so that we can cite the reference in this datasheet.

    ab126704 has been referenced in 9 publications.

    • Ge C  et al. Nrf2 deficiency aggravates PM2.5-induced cardiomyopathy by enhancing oxidative stress, fibrosis and inflammation via RIPK3-regulated mitochondrial disorder. Aging (Albany NY) 12:4836-4865 (2020). PubMed: 32182211
    • Song CH  et al. Effects of 17ß-estradiol on colorectal cancer development after azoxymethane/dextran sulfate sodium treatment of ovariectomized mice. Biochem Pharmacol 164:139-151 (2019). PubMed: 30981879
    • Parsanathan R & Jain SK Hydrogen sulfide increases glutathione biosynthesis, and glucose uptake and utilisation in C2C12 mouse myotubes. Free Radic Res 52:288-303 (2018). PubMed: 29378451
    • Qiu L  et al. A Naturally-Occurring Dominant-Negative Inhibitor of Keap1 Competitively against Its Negative Regulation of Nrf2. Int J Mol Sci 19:N/A (2018). PubMed: 30042301
    • Qiu L  et al. Oncogenic Activation of Nrf2, Though as a Master Antioxidant Transcription Factor, Liberated by Specific Knockout of the Full-Length Nrf1a that Acts as a Dominant Tumor Repressor. Cancers (Basel) 10:N/A (2018). PubMed: 30562963
    • Sun W  et al. Proanthocyanidins Attenuation of H2O2-Induced Oxidative Damage in Tendon-Derived Stem Cells via Upregulating Nrf-2 Signaling Pathway. Biomed Res Int 2017:7529104 (2017). PubMed: 29201913
    • Bakshi R  et al. Neuroprotective effects of urate are mediated by augmenting astrocytic glutathione synthesis and release. Neurobiol Dis 82:574-579 (2015). PubMed: 26341543
    • Zhang J  et al. Comparisons of ethanol extracts of chinese propolis (poplar type) and poplar gums based on the antioxidant activities and molecular mechanism. Evid Based Complement Alternat Med 2015:307594 (2015). Mouse . PubMed: 25802536
    • Jain SK  et al. Hydrogen sulfide upregulates glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione and inhibits interleukin-1ß secretion in monocytes exposed to high glucose levels. Metab Syndr Relat Disord 12:299-302 (2014). WB ; Human . PubMed: 24665821

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    1-2 of 2 Abreviews or Q&A

    Western blot abreview for Anti-GCLM antibody [EPR6667]

     Good
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Mouse Tissue lysate - whole (Liver lysate)
    Gel Running Conditions
    Reduced Denaturing (4-12% gel)
    Loading amount
    10 µg
    Specification
    Liver lysate
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 20 2018

    Western blot abreview for Anti-GCLM antibody [EPR6667]

     Excellent
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (RPE)
    Gel Running Conditions
    Reduced Denaturing (4-12)
    Loading amount
    20 µg
    Specification
    RPE
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Abcam user community

    Verified customer

    Submitted Nov 09 2018

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